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Activity and expression of adenosine diphosphate glucose pyrophosphorylase in

Activity and expression of adenosine diphosphate glucose pyrophosphorylase in developing rice grains:Varietal differences and implications on grain ?lling

T.Anjana Devi a ,N.Sarla b ,E.A.Siddiq b ,1,Ravi Sirdeshmukh a ,*

a Centre for Cellular and Molecular Biology,Council of Scienti?c and Industrial Research,Uppal Road,Habshiguda,Hyderabad 500007,AP,India b

Directorate of Rice Research,Rajendranagar,Hyderabad,AP,India

1.Introduction

Starch accumulation during grain ?lling in cereals may be controlled by the activities of enzymes of starch synthesis and/or partitioning of the metabolites for starch synthesis [1–4].Among the enzymes involved,ADP-glucose pyrophosphorylase (AGPase;EC 2.7.7.27)catalyzes the ?rst committed step in starch biosynthesis in plants—the conversion of glucose-1-phosphate and ATP into ADP-glucose (the precursor of starch)and pyrophosphate (PPi).Transgenic studies in potato tubers [5],wheat [6],maize [7]as well as rice [8,9]demonstrate changes in grain ?lling and starch yield by altering the activity of AGPase supporting the conclusion that AGPase is a key regulatory enzyme for starch biosynthesis [10].Plant AGPases are hetero-tetramers of two identical small subunits (SS)and two identical

large subunits (LS)(see Ref.[11],for review).The small subunits are mainly responsible for the catalytic properties whereas the large subunits are of regulatory importance.The synthesis of ADP-Glu occurs within the plastids in many tissues examined.However,in rice endosperm (Oryza sativa)and other crop plants,ADP-Glu is also made outside the plastid,presumably in the cytosol by another AGPase which may account for most (85–95%)of the total endosperm activity [12–14].Interestingly,the two seed-speci?c isoenzymes of the large subunit are devoid of a plastidial targeting sequence,which is in agreement with the localization of AGPase activity mainly in the cytosol of the graminaceous endosperm.The SS and LS of AGPase in plants are encoded by multiple genes—two for SSs and four for LSs,expressed in a tissue-speci?c and developmentally regulated manner [2].Evolutionarily,the small subunit of AGPase is much more conserved relative to the large subunit.This difference is due to greater evolutionary constraints on the small subunit.Both the subunits are however equally predisposed to activity altering mutations [15].

AGPase activity changes may be a result of differences in the (a)gene sequence,(b)changes in its expression [6,8,16,17],(c)the allosteric controls at the level of the activity [2,18],or (d)protein

Plant Science 178(2010)123–129

A R T I C L E I N F O Article history:

Received 1August 2009

Received in revised form 20October 2009Accepted 24October 2009

Available online 2November 2009Keywords:

ADP-glucose pyrophosphorylase Regulation Grain ?lling Rice

Starch synthesis

A B S T R A C T

ADP-glucose pyrophosphorylase (AGPase)is a key enzyme in starch biosynthesis in plants.The activity,expression and gene sequence of AGPase were studied in grains of ?ve rice cultivars representing different origins to gain insight into their variations in relation to starch accumulation.The varieties were Jaya,GEB24(indica),BSI 115(japonica),Mahsuri (indica-japonica derivative)and CoRH2(hybrid).The grain weight increased in all of them until 20dpa,although at varying rates.It was relatively steady in Mahsuri,in others there was a steep increase from 10to 20days post-anthesis (dpa).The total AGPase activity increased steadily from 5to 20dpa in Jaya and GEB24,but dropped after 15dpa in BSI 115and CoRH2while in Mahsuri,it maintained the 10dpa low level until 20dpa.The AGPase cDNAs did not show any sequence differences in the ?ve varieties.The transcripts of both sub units of AGPase accumulated comparably during the initial phase of grain development suggesting their coordinated expression and dropped after 15dpa in all the varieties.On the other hand,AGPase protein level remained unchanged after reaching the maximum in CoRH2and continued to increase up to 20dpa in the other four varieties.Thus the drop in the enzyme activity cannot be attributed to changes in the transcript or protein amounts per se.We infer that the drop in activity,its varying levels and varying duration in the varieties studied is likely due to differential stability of the active enzyme protein during grain development and that the extended duration of enzyme activity may contribute to increased grain weight.

Abbreviations:dpa,days post-anthesis;AGPase,ADP-glucose pyrophosphorylase;SS,small subunit;LS,large subunit.

*Corresponding author.Tel.:+914027192562;fax:+914027160591/310.E-mail address:ravi@ccmb.res.in (R.Sirdeshmukh).1

Present address:BioTechnology Unit,ANGR Agricultural University,Rajen-dranagar,Hyderabad 500030,India.

Contents lists available at ScienceDirect

Plant Science

j o u r n a l ho m e p a g e :w w w.e l s e vi e r.c o m /l o c a t e /p l a n t s c i

modi?cations[2,19].Some recent reports also implicate thermal stability of the enzyme to be a regulatory mechanism[20]. Although AGPase is considered to be a key enzyme[8],AGPase regulation and the underlying mechanisms in relation to grain ?lling in rice are not completely understood and investigating them will help in getting further insights into the process of starch accumulation during grain?lling.Availability of diverse rice varieties with varying patterns of starch content,grain size and grain?lling offers a useful system to explore the biochemical and regulatory aspects of AGPase.We therefore undertook the study of AGPase expression and activity in select rice varieties of different origins,during grain?lling.For this,we studied the changes in the enzyme activity,its transcript and protein levels at5,10,15and20 days post-anthesis as well as the gene sequence of AGPase in?ve rice varieties-Jaya,GEB24(indica),BSI115(japonica),Mahsuri (indica-japonica derivative)and CoRH2(hybrid).We?rst describe the pattern of grain weight increase in these varieties during grain development.Second,we present analysis of the patterns of AGPase activity,AGPase protein levels,transcript levels and the cDNA sequence of both small and large sub units.From the results, we conclude that the stability of AGPase protein varies in varieties resulting in different durations of enzyme activity during grain development which may be the basis of grain weight differences.

2.Materials and methods

2.1.Choice of the rice cultivars

Rice cultivars for the present study were selected on the basis of their genetic differences/origin,i.e.indica,japonica,indica-japonica derivatives and hybrid.These?ve rice varieties—Jaya, GEB24(indica),BSI115(japonica),Mahsuri(indica-japonica derivative)and CoRH2(hybrid)also differ in the proportion of ?lled grains and size of the grains.Table1presents the salient features of these rice varieties[21].

2.2.Plant material

Rice varieties selected for the study were obtained from the Directorate of Rice Research,Hyderabad,India.These were grown in a glasshouse and growth conditions were maintained as16h light(day)/8h dark(night)for all the varieties.

The sampling of the developing grains was done following the scheme of Counce et al.[22].The spikelets on the main panicle were tagged on the day of anthesis.Sampling was done at5,10,15 and20days post-anthesis(dpa).The grains were immediately stored in a cryo system maintained atà708C.2.3.Determination of grain weight

Hundred milligram of grains at four stages of development(at5, 10,15and20dpa)from each of the rice varieties were weighed using digital weighing balance,the number of grains in the sample counted and the weight of a single grain was calculated.For dry weight,grains were dried at37*C and their weight followed at intervals up to48h,until constant value.

2.4.Protein extraction,quantitation

Crude protein extracts of the developing grains,were prepared by rapidly homogenizing approximately100mg of the grains using a mortar and pestle in1ml protein extraction buffer containing50mM Tris–HCl,pH7.4,1mM EDTA,2mM MgCl2, 2mM DTT,2.5mM PMSF,0.1%Triton-X-100[23].The homogenate was centrifuged at12,000rpm for10min,the supernatant was collected and the protein concentration of rice grain protein extracts was determined using the Bradford protein assay kit (Biorad,Hercules,CA,USA)according to the manufacturer’s instructions and using BSA as a standard.This extract was used for studying AGPase activity in grains from each rice variety during development(5,10,15and20dpa).

2.5.Enzyme assay

AGPase activity was measured in the direction of ADP-Glu synthesis at378C essentially according to the procedure described by Ghosh and Preiss[24].The standard assay mixture,200m l, contained100mM Tris,pH7.5,2.5mM MgCl2,0.5mM3-PGA,14C-labeled glucose-1-p(1?105cpm)(New England Nuclear,Boston, MA,USA),0.5mM unlabelled glucose-1-P,5mM ATP,0.5mg BSA. The reaction was initiated by the addition of known amount of the crude grain protein extract and was allowed to proceed for10min, at378C.After10min,the reaction was stopped by boiling the reaction mixture for45s.The reaction mixture was treated with alkaline phosphatase to destroy unreacted glucose-1-P and immediately chromatographed in descending direction in 2cm?18cm DE81paper strips,but using double distilled water as solvent(based on pilot studies).ADP-14C glucose(remaining at the origin)was determined by Packard Tri carb1500liquid scintillation counter(Packard,Downers Grove,IL,USA)using toluene as solvent with PPO and POPOP(Diphenyl oxazole and Bis diphenyl oxazole)as scintillators.The amount of the reaction product formed,in nmoles per min,was calculated.Under these optimized assay conditions the activity was linear with respect to time and amount of extract added,as studied in pilot experiments.

Table1

Rice varieties selected for the study and their salient features.

S.no.Cultivar Type Height Days of maturation Grain

weight/1000

grains in grams

Salient features

1.Jaya Indica Semi-dwarf

90cm Medium-maturing

135days

25TN1/T141,derivative high-yielding;

photoinsensitive,wide adaptability

2.GEB-24(Government

Economic-Botanist)Traditional indica Tall120cm Late maturing

150days

18Developed at Coimbatore;Spontaneous

mutant of Konamani,photo-sensitive;

known for its grain quality;all grains?lled

3.BSI115

(Bulu Seed Increase)Tropical japonica

(Javanica)

Medium tall

110cm

Medium-maturing

135days

30Designated IR66159-189-5-5-3by IRRI,

long and heavy panicles;medium tillering

4.Mahsuri Indica-japonica

derivative Semi tall

110cm

Medium late

maturing>140days

15Developed in Malaysia;derivative of

MayangEbos80/Taichung65,medium

slender grain;weekly photo-sensitive,

high photosynthetic ef?ciency

5.CoRH2(Coimbatore

Rice Hybrid Hybrid Semi-dwarf

90cm

Medium early

maturing125days

23IR58025A/C20R,derivative high-yielding,

high tillering ability,photo insensitive T.A.Devi et al./Plant Science178(2010)123–129

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All assays were performed in triplicates and readings were reproducible within 10%error.2.6.AGPase gene sequencing

To get cDNA fragments encoding AGPase small and large subunit genes,total RNA was isolated from the grains of the rice varieties using the procedure of Zeng and Yang [25].Two micrograms of total RNA was reverse transcribed using a reverse transcription kit (Promega Corporation,Madison,WI,USA)with an oligo dT primer according to the manufacturer’s https://www.wendangku.net/doc/7517735380.html,ing the Primer3.0software,two sets of primers were designed for ampli?cation of AGPase small and large subunit cDNAs based on the Oryza sativa AGPase cDNA sequences from the NCBI database (Accession number AY028315for AGPase SS,AY028314for AGPase LS)and were synthesized in CCMB in-house facility (Table 2).RT-PCR reactions were carried out as per the conditions described in Table 3,and the reaction products were separated using 1%(w/v)agarose gels by electrophoresis along with the positive and negative PCR controls.2.7.Sequence analysis

All the RT-PCR products from the varieties were sequenced and compared with the corresponding AGPase cDNA sequences encoding SS and LS from the NCBI database.Sequencing was performed on a ABI 377DNA sequencer (Perkin-Elmer,Foster City,CA,USA)using cyclic sequencing with a dye terminator as recommended by the manufacturer.All PCR products were puri?ed using a QIA quick gel extraction kit (Qiagen,Hilden,Germany)before each sequencing reaction.2.8.Transcript analysis

Semiquantitative RT-PCR was performed to study transcript levels of AGPase small and large subunits of each rice variety during grain development.For this,RT was performed as described above.PCR conditions were optimized to ensure product intensity within the linear phase of ampli?cation.The house keeping gene actin was selected as the internal control to normalize the differences in sample concentration and loading.The size of the expected product was determined by electrophoresis of reaction products in agarose gels along with the 1kb ladder.The product

obtained was 1kb long for small subunit (using the primers SSBI and SSPIII)and 800bp long for large subunit (using the primers ALSFI and ALSRI)of AGPase.AGPase transcript levels between and within the varieties were compared by densitometric scanning of the gel bands and the values expressed as gene:actin ratio.2.9.SDS-PAGE and immunoblotting for determination of AGPase protein

Rice grain crude protein extracts were mixed with SDS gel loading buffer,heated to 1008C for 3min,loaded on 10%SDS-PAGE gels and electrophoresis carried out.The apparent molecular weights of polypeptides on the SDS-gel were determined using prestained protein markers (Bangalore Genei,Bangalore,India)in the electrophoresis run.The proteins from the SDS gels were electroblotted onto Hybond-P membrane (Amersham Life science,Buckinghamshire,UK).An antigenic synthetic peptide (19amino acid residues,RPFVFTPRAVSDSRSSQTC)of AGPase small subunit was selected and chemically synthesized by Alpha diagnostic international (San Antonio,CA,USA).Antibodies were raised against this synthetic peptide in rabbit.Following electroblotting,the membrane was blocked with 2%(w/v)bovine serum albumin in Tris buffered saline Tween 20(TBST)and exposed to the above antibodies (diluted to 1:1000,pre determined in pilot experiments for least cross reactivity).Immunodetection was performed using goat anti-rabbit secondary antibody conjugated with horseradish peroxidase using the DAB reaction kit (Bangalore Genei,Bangalore,India).3.Results

3.1.Pattern of grain weight of the rice varieties during grain development

We determined fresh and dry weight of the developing grains from all varieties as described in Section 2.The weights reached maximum by 20dpa and were the highest for Jaya and BSI 115,the least for Mahsuri,whereas for GEB 24and CoRH2they were intermediate (Fig.1A and B).In general,all the varieties showed comparable and gradual increase in dry weight until 20dpa except in the case of BSI 115and Jaya.BSI 115showed a striking increase between 10and 15dpa and between 15and 20dpa.In Jaya,the steep increase was observed between 15and 20dpa reaching

Table 3

PCR conditions used for ampli?cation of small and large subunits of AGPase gene.AGPase gene Primers

PCR conditions AGPase SS GPSF2(F)SSP2(R)Step 1:948C-2min;Step 2:948C-1min,518C-1min,728C-2min (34times);Step 3:728C-10min;Step 4:48C hold AGPase SS SSB1(F)SSP3(R)Step 1:948C-2min;Step 2:948C-40s,578C-40s,728C-2min (34times);Step 3:728C-10min;Step 4:48C hold AGPase LS ALSF1(F)ALSR1(R)Step 1:94o C-2min;Step 2:94o C-40s,57o C-40s,72o C-2min (34times);Step 3:72o C-10min;Step 4:48C hold

AGPase

ALSF2(F)ALSR2(R)

Step

94o C-2min;Step 2:94o C-1min,51.6o C-1min,72o C-2min (34times);Step 3:72o C-10min;Step 4:48C hold

SS:small subunit;LS:large subunit;F:forward primer;R:reverse primer.

Table 2

Oligonucleotide primers used for AGPase gene sequencing and RT-PCR https://www.wendangku.net/doc/7517735380.html, of the primer Sequence Start End AGPase small subunit GPSF2(F)50GTCAGTACTCCGGCCACCTCTC 30

7697SSP3(F)50CAGGGTACTGCAGATGCTGTGCGAC 30613636SSP2(R)50GTCGCACAGCATCTGCAGTACCCTG 30637613SSB1(R)

50CGCCTAGGGGTTGCCTTTATATGACTG 3016331607AGPase large subunit ALSF1(F)50AACACT TGG GCAGGTTTGTC 30

1535ALSF2(F)50CCAGAGGGTGCTGATTTGGAATCGATG 30853880ALSR1(R)50CATCGATTCCAAATCAGCACCCTCTGG 30880853ALSR2(R)

CTGAAGCTTCTATTATATGATCGGCCCG 30

1620

1592

Start and end indicate the start and end positions of the primers on the reference sequences AY028315for small subunit and AY028314for large subunit of the NCBI database.

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levels comparable to BSI 115by 20dpa.The dry weight re?ected the seed size in these varieties.BSI 115,a japonica variety has seeds with low l/b ratio with high dry weight.Jaya,an indica variety,had the next highest grain weight.Mahsuri,a medium slender grain variety,had the minimum dry weight.

3.2.Pattern of AGPase activity in rice varieties during grain development

AGPase activity pro?les varied among the ?ve varieties studied.The activity was low at 5dpa in all varieties (Fig.1C),and peaked by 15dpa in BSI 115,Mahsuri and CoRH2and then dropped except in Mahsuri where the low 10dpa level stayed more or less unchanged till 20dpa.Interestingly,in Jaya and GEB 24it continued to increase until 20dpa.Among the ?ve varieties,the peak activity was the highest in GEB 24followed by Jaya—a popular high-yielding variety (Indica),BSI 115(Japonica),CoRH2(a hybrid)

and Mahsuri (an indica-japonica derivative)and ranged from approximately 2nmoles ADP Glu/min/grain in Mahsuri to 9nmoles ADP-Glu/min/per grain in GEB 24(Fig.1).On derivation,this would be 70–140nmol ADP-glu/min/mg protein (details not shown)comparable with 75–123nmol ADP-Glu/min/mg protein reported by Smidansky et al.[8].It is interesting to note that GEB24a variety well known for all ?lled spikelets and Jaya—also known for good grain ?lling,exhibited high AGPase activity until 20dpa.In contrast,in the hybrid CoRH2,known to have high number of spikelets but a signi?cant number being un?lled,the AGPase activity was lower and dropped after 15dpa.In Mahsuri,the overall level of activity was the lowest but maintained at about the same level from 10to 20dpa.Thus although the AGPase activity pro?les varied among the varieties studied,overall they indicated good correlation with grain ?lling/grain weight consistent with earlier report of Mohapatra et al.[30]that the activities of AGpase (and sucrose synthase)were higher in a big sized seed compared to a small sized seed.

3.3.AGPase gene sequence determination

Multiple,tissue-speci?c forms of the SS and LS of AGPase have been shown to exist in plants,encoded by multiple genes—two for SS and four for LS,expressed in a tissue-speci?c and developmen-tally regulated manner [26,27].The SS genes are detected both in leaf and seed while two LS genes are detected in seed and one exclusively in leaf.With an aim to relate AGPase activity differences with its gene sequences we determined nucleotide sequences of the AGPase cDNAs from these varieties and carried out homology comparisons with the AGPase sequences available in the NCBI database representing complete ORFs.There were six such complete sequence entries in the NCBI database and our sequences revealed highest homology with AGPase sequences AY028315(small sub unit,AGPS2)and AY028314(large sub unit AGPL1).Further the cDNA sequences of the small subunits (SS)of all ?ve rice varieties were identical.The cDNA sequences of large subunit (LS)were also identical except in case of GEB24,which showed a difference at two nucleotide positions (156and 273position of the AGPase LS reference sequence AY028314).Since these nucleotide differences were located at the position of the wobble base,it did not result in any change at amino acid level in the deduced protein sequences.

3.4.AGPase mRNA expression in rice grains

Since AGPase cDNA sequences could not explain the variations in AGPase activity in the rice varieties.AGPase transcript levels and AGPase protein levels were determined at four stages (5,10,15and 20dpa)during grain development.To examine the expression pattern of AGPase SS and LS mRNA in developing grains,total RNA was isolated from grains collected at 5,10,15and 20dpa and subjected to semiquantitative RT-PCR analysis using the primers SSBI and SSPIII for small subunit and ALSF1and ALSR1for large subunit,respectively (Table 2).The levels of AGPase mRNA differed during the grain ?lling period,in the varieties studied.It was low at 5dpa,but gradually increased reaching a peak at 15dpa followed by decrease at 20dpa,in all the ?ve varieties (Fig.2).The maximum level reached in each was 2–3-fold higher in compar-ison to the level at 5dpa.As a control,the expression of the house keeping gene actin was studied and was found to stay relatively unchanged in all these rice varieties.The period between 5and 15dpa is the time when the grains start to transcribe the gene and make the protein to initiate starch synthesis and accumulation.The drop at later point may be due to feedback regulation of AGPase transcription in response to the level of ADP-Glu formed [28].The results indicate that the two subunit mRNAs

accumulate

Fig.1.Fresh/wet grain weight (A),dry grain weight (B)and AGPase activity (C)during grain development in each rice variety.The rice grains were obtained at four different stages of grain development from each rice variety as described under Section 2.The grain weights were determined using a digital weighing balance and expressed as mg per grain.AGPase activity in the endosperm protein extracts of the developing grains was determined by measuring the amount of ADP-Glu synthesized as described in Section 2.The activity as nanomoles of ADP-Glu formed per min per mg of protein was calculated in terms of activity per grain on the basis of the number of grains used for protein extraction.The developmental stage of the grains is shown as 5dpa,10dpa,15dpa and 20dpa which indicate days post-anthesis.

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at a comparable rate suggesting their coordinated expression during grain development which is important for AGPase regulation as discussed in earlier reports [25,30].Their overall pro?le during grain development overlaps with the AGPase activity changes in varieties except in Jaya and GEB 24.3.5.AGPase protein levels

We further studied AGPase protein expression in an attempt to relate it to the activity patterns in varieties.Since the transcript analysis re?ected coordinated expression of the two subunits,we studied the level of only small subunit of AGPase in various varieties as a representation of total AGPase level.The immunoblot analysis using antisera against antigenic peptide (synthetic)of small subunit of rice AGPase is shown in Fig.3.The protein pro?les indicate a gradual increase in the protein levels up to 20dpa in four varieties.In GEB 24,the maximum accumulation of the protein occurred at 5dpa itself which remained more or less unchanged until 20dpa.The observation that AGPase activity pro?le increased in Jaya and GEB24during grain ?lling period,despite more or less unchanged trend in protein levels indicates its high stability compared to that of other varieties.Thus there appear to be

variations in the structural or conformational stability of the protein affecting its activity during grain development.4.Discussion

Grain development and ?lling is a complex process involving many factors at the individual grain level as well as at the plant level.In maize it has been shown that seed weight is dependent on the alleles of endosperm AGPase [16,17].In other studies using transgenics,it is reported that increased AGPase activity stimulates additional seed set increasing seed weight/plant but not starch content of individual seeds [8].Our ms.represents an effort to correlate AGPase activity and grain wt variations during grain development,using the varieties of different origins.The study contributes three important insights:

There is natural variation in the ‘levels’of AGPase activity as well as AGPase stable activity period during grain development,plausibly contributing to grain weight differences among the varieties.

Sustained grain weight increase even after drop in AGPase activity during grain development,indicating presence of adequate residual ADP-Glu and its utilization by other

enzymes

Fig.2.Expression pattern of the small subunit (A)and large subunit (B)of AGPase in developing rice grains by semiquantitative RT PCR analysis.RT PCR reactions were carried out using SS and LS speci?c primers,as detailed in Section 2.Relative intensity of AGPase SS and LS cDNA (C and D)in different varieties with reference to the intensity of actin cDNA,were quantitated by densitometric scanning of EtBr stained agarose gels of the RT PCR reaction products and plotted.The results represent mean of two independent experiments.The developmental stage of the grains is shown as 5dpa,10dpa,15dpa and 20dpa,which indicate days post-anthesis.

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of the starch synthesis pathway,independent of concurrent AGPase activity.

At the regulation level,the study indicates that the variations in AGPase activity in rice are not attributable in terms of the gene sequences or the transcript levels alone but the regulation of AGPase activity may possibly occur at the level of protein stability.

Together this and the information from other independent study designs in rice,would be useful to further understand the natural relationship between AGPase expression,activity and grain ?lling/grain weight in rice.

4.1.AGPase activity and grain weight in rice varieties

Murchie et al.[29]reported that rapid grain ?lling phase occurred approximately 10dpa in most varieties they studied,i.e.,?ve new plant types (NPT)(tropical japonica)and one indica variety.In transgenic of maize AGPase [7]and in tropical japonica rice M202[8]also,the maximum activity level was observed at 10dpa.In our study,the activity was found to increase from 5to 15dpa and then dropped in three varieties—BSI 115(tropical japonica),CoRH2(a hybrid),consistent with earlier observation with cereal grains in general [31,32].In Mahsuri (indica-japonica derivative),AGPase activity which reached maximum at 10dpa dropped only marginally;it maintained virtually unchanged up to 20days.However,it was the lowest seen of all varieties studied.Notably,in the other two varieties studied by us—Jaya and GEB24,the activity curve steadily increased until 20dpa.As shown in Fig.1,grain weight on the other hand continued to increase until 20dpa in all,the highest increment occurring between 15and 20dpa.Mahsuri,showed relatively slow rate of grain weight increase.The increase in AGPase activity was also maximum between 5and 15dpa in all the varieties.The activity levels in Mahsuri and CoRH2and the activity drop in the latter explains low

grain weight was also low in the two.This may also explain high percentage (up to 40%)of un?lled grains in CoRH2[33,34].In Jaya and GEB24,the AGPase activity also increased up to 20dpa.This probably correlates with the higher grain weight and also grain ?lling in these two important varieties.

4.2.ADP-Glu availability and utilization under low AGPase activity It is notable that,the grain weight was the highest in BSI 115although the activity was not and it also dropped after 15dpa,both in BSI 115and CoRH2.With wild type and four transgenic CS8rice lines expressing 3–6-fold higher AGPase activity,Nagai et al.[4]observed elevated levels of ADP-Glu but not comparable increase in seed starch suggesting accumulation of ADP-Glu.We have not measured ADP-Glu,if accumulated,but our results with BSI 115and CoRH2provide circumstantial support to infer that concurrent synthesis of ADP-Glu may not be essential for the reactions of the subsequent enzymes in starch-biosynthesis pathway,if adequate amounts of residual ADP-Glu is available to be utilized by these enzymes even after AGPase activity reduces signi?cantly.However,the duration of stable AGPase activity period may be an important determinant for grain ?lling and/or grain weight and this is consistent with the observation that Jaya and GEB 24(high activity up to 20dpa)have higher grain weight and good grain ?lling as compared to Mahsuri and CoRH2((low activity and stable activity period up to 15dpa))which have low grain weight (Mahsuri)or have large proportion of un?lled grains CoRH2(see Section 3).In BSI 115,although the duration of stable AGPase activity does not extend up to 20days,the level is very high and might result in high grain weight.4.3.What is the basis of longer period of AGPase activity during grain ?lling?

cDNA sequencing of AGPase small and large subunit of these varieties showed almost complete identity and could not explain the activity variations among varieties.Coding sequence dictates the structural features of the protein but promoter region cis elements may in?uence ef?ciency of transcription and the levels of the transcript.Transcript levels are known to generally correlate with AGPase activity pro?le as reported in several plant species [2].We therefore studied levels of AGPase transcripts and the protein during grain development.In general,the transcript levels increased until 15dpa and dropped after that in all the varieties and were higher for Jaya and more or less comparable for all the remaining varieties.Their pro?le correlated in general with the AGPase activity pattern in all varieties except in Jaya and GEB24,in which regardless of the drop in mRNA levels after 15dpa,the protein levels and the activity continued to increase.Further,AGPase protein levels in Jaya and BSI 115were comparable (highest of all varieties in BSI 115),even if the mRNA levels were not.These observations suggest following points:(a)virtually identical pro?les of both SS and LS transcripts are consistent with the coordinated expression of the two subunits of AGPase during grain development.b.general abundance or excess of translatable AGPase mRNA so that the steady state level of the protein is maintained even when the total mRNA levels drop,and (b)the AGPase activity varies regardless of comparable levels of AGPase protein.The drop in the AGPase activities in three varieties (BSI 115,CoRH2and Mahsuri)despite comparable protein levels during grain development may suggest conformational destability of the enzyme,as against greater stability of the activity of the protein in the other two varieties Jaya and GEB 24.The basis of this is not clear.In view of the use of protease inhibitors during protein extraction or assays with freshly prepared extracts,it is unlikely that differences arise during the in vitro experimental conditions used.In addition,all the assays were carried out in the presence

Fig.3.AGPase small subunit protein levels during grain development in each rice variety.Protein was extracted from grains at various stages of grain development from each rice variety.A total of 10m g protein was run on SDS-PAGE (A),transferred onto nitrocellulose membrane and reacted using antibodies against small subunit of AGPase and the immunoreactive band was identi?ed (B).The densitometric quantitation of the immunoreactive band was carried out to assess AGPase (small subunit)protein levels and the values expressed as arbitrary units,were plotted as bar diagrams (C).The developmental stage of the grains is shown as 5dpa,10dpa,15dpa and 20dpa which indicate days post-anthesis.

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equal and excess amounts of allosteric effectors—PGA or Pi[2,4].As discussed,it is more likely that the differential activity patterns are determined by the conformational status of AGPase protein.Either the subunit proteins do not fold properly to form the tetrameric active enzyme and/or the tetramers are not stable and get disaggregated.The overall microenvironment of the developing grain collectively dictated by the nature of starch,its structure, global grain protein pro?les[35],pH and redox potential of the grain may be an important determinant for this.

Regardless of the exact mechanism,the AGPase stability and activity for longer durations during grain development as well as increasing activity observed in the two varieties—Jaya and GEB24 late in the development may contribute to higher grain weight in these varieties.Although not yet proven as a cause/effect relationship,the overall correlation between grain weight and AGPase activity holds true for all varieties in the initial stages, while the data with Jaya and GEB24point to a correlation even during late period of grain development.This natural variation observed in AGPase activity pro?le during grain?lling is an important component combined with the role of other enzymes in starch synthesis as discussed in[30].It would be interesting to design breeding experiments to explore if high AGPase activity late in grain?lling stage cosegregates with enhanced seed weight in a population segregating for grain weight so that cause/effect relationship could be established

Acknowledgements

This work was undertaken under the National Professorship grant to EAS from Indian Council of Agricultural Research, Government of India and under the Network Program on Functional Genomics in rice‘‘Identi?cation and functional analysis of genes related to yield in Rice’’supported by the Department of Biotechnology,Government of India.Anjana Devi was a recipient of Research Fellowship from the Council of Scienti?c and Industrial Research.Help from Dr.MRV Prasada Reddy in initiation of the project is greatly appreciated.

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那些好看的流程图是用什么软件画出来的

那些好看的流程图是用什么软件画出来的导语：我们经常会在商业场合下，看到一些非常精美的PPT，有的还自带流程图。小伙伴们是否也特别希望可以制作出一样效果的流程图PPT？其实，只要找对一款流程图软件就能够轻松实现。来看下本文的教程介绍！免费获取亿图图示软件：https://www.wendangku.net/doc/7517735380.html,/edrawmax/ 怎么画出好看的流程图？看似专业又好看的流程图当然是用专业的软件画的了。亿图图示，一款可以绘制专业流程图的工具，拖拽式操作，10000+矢量素材，支持导出PPT、图片、PDF、HTML、PS等格式。除此以外，还可以将流程图作品存储到亿图云，或直接打印出来。

亿图图示软件特色： 1、来自全球超过600万的用户选择下载安装。 2、支持多系统操作：亿图图示工作流程图图可以在Windows，Mac 和 Linux上进行制作。 3、产品升级：亿图软件不断更新升级，重视用户体验度。 4、简单操作：一键式绘制工具帮助用户绘制快捷，方便使用者管理工作项目。如何在PPT里画出好看的流程图使用亿图图示软件绘制流程图，可以通过软件的导出功能，一键将文件导出为PPT格式的文件。导出的文件，可以在PPT中继续编辑、修改，进行幻灯片演示等等。

所谓千言万语不如一张图，尤其是在进行PPT幻灯片演示的时候，将繁杂琐碎的事物用简单、明了的流程图表达出来，能够让观看者对事物进行中的顺序、布置和安排有更加生动的了解。下面让我们一起来看几张适合做PPT展示用的流程图模板：圆点PPT流程图模板

扁平化商务总结汇报PPT模板极简设计风的PPT流程图模板

page的用法总结大全

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如何使用网络分析仪德力网络分析仪NA7682A NA7682A矢量网络分析仪吸取了前几代和国内外各款网络分析仪使用的经验，结合了最新国际仪器发展的技术和态势，是Deviser德力仪器最新推出的第四代矢量网络分析仪,作为国内主流的网络分析仪,下面介绍网络分析仪的使用技巧如下。频率范围从100kHz到8.5GHz频段，为无线通信、广播电视、汽车电子、半导体和医疗器件等行业射频器件、组件的研发和生产的使用提供了高效、灵活的测试手段，进入了民品、工业、科研教育和军工等领域。其主要的特点是和主流网络分析仪是德的E507X系列指标和指令上做到兼容，在客户使用的性价比上非常优秀的选择。在射频器件、基站天线、手机天线、GPS天线等、通信系统模块分析等领域成功的测试经验使越来越多的客户开始使用这款网络分析仪，在低频、800/900M、1800/1900M、2100M、5G/5.8G等的产品频率使用领域内广泛使用。深圳市良源通科技有限公司专业服务和销售射频和通信仪表多年，是德力仪器国内最重要的合作伙伴和一级代理商，结合自己多年的技术积累和客户使用的配合测试，得到丰富经验。在仪器的售前和售后服务上面具有自己的优势。提供大量仪器试用和使用方案的设计，给客户在设备开发、产品研制和批量生产上都提供方便和最有优势的选择。产品特点： 1、12.1英寸1280*800 TFT触摸屏 2、频率覆盖范围: 100 kHz 至 8.5 GHz 3、阻抗：50Ω 4、动态范围: >125 dB （比E5071C宽7-12dB) 5、极低的迹线噪声: <0.005 dBrms (在 3 kHz IFBW) 6、快速的测量速度: 80usec/点 7、分析和误差修正和校准功能 8、通过USB、LAN 和 GPIB 接口进行系统互联 9、时域分析（选件）：时域传输、反射特性分析；距离上的故障定位。 10、数据变换：涉及多种形式的阻抗、导纳变换。 11、滤波器分析：自动分析出：插损、3dB带宽、6dB带宽、带内纹波、带外抑制、Q值、矩形系数

教你如何利用WORD制作漂亮的流程图

教你如何利用WORD制作漂亮的流程图在日常的很多实际任务中，我们可能需要表达某个工作的过程或流程。有些工作的过程比较复杂，如果你仅仅用文字表达，通常是很难描述清楚的。与此同时，听者也难于搞懂，在这种情况下，最好的方式就是绘制工作流程图，图形的直观性会让双方都大大获益。笔者曾经介绍过用MS VISIO 2002制作工作流程图的方法（请参阅《用Visio做工作流程图》），但是很多朋友反映自己的电脑上并没有安装VISIO，而且对VISIO还有种陌生感。为了便于更多的朋友轻松地制作工作流程图，下面我们介绍利用Word制作漂亮工作流程图的方法。任务要求：这里我们还是以制作毕业论文写作流程图为例，该工作流程图表达的是数理信息学院毕业生毕业论文的制作过程。为了让朋友们对制作目标有个大概的了解，下面先来看一下完成后的效果图(图 1)。制作共包括以下步骤：步骤一：页面和段落的设置步骤二：制作流程图的标题步骤三：绘制流程图框架步骤四：流程图的对齐与修饰步骤五：添加连接箭头步骤六：添加折线连接符和说明性文字为了提高工作流程图的制作效率，在具体制作之前应该先在头脑中构思一下流程图的大概效果，最好能够在稿纸上把效果图画出来，这往往比边想边做要快很多。在纸上画好草图之后，我们就可以打开Word 2003进行具体的制作了，本例的操作是在Word 2003中进行的，使用Word其它版本的朋友可以参照进行。步骤一：页面和段落的设置为了流程图有较大的绘制空间，我们先来设置一下页面。 1．启动Word 2003，打开一个空白文档，并切换到页面视图。选择菜单“文件→页面设置”命令，打开“页面设置”对话框，在“页边距”选项卡中，设置上下边距为“1厘米”，左右边距为“2厘米”(图 2)，完成后单击“确定”按钮。

Seated的用法小结

Seated的用法小结 seated是一个比较特别的过去分词，说它特殊一是因为它的词性尚有不确定性——它有时是过去分词，有时又具有形容词的性质，像是一个形容词；二是因为这样一个很少引人注意的过去分词，在近几年的高考英语考题中经常“露脸”，一下子变成了一个热点词汇。下面我们先来看几道高考题： 1. Please remain __________ until the plane has come to a complete stop. (山东卷) A. to seat B. to be seated C. seating D. seated 2. Please remain __________; the winner of the prize will be announced soon. (辽宁卷) A. seating B. seated C. to seat D. to be seated 3. Can those _________ at the back of the classroom hear me? (福建卷) A. seat B. sit C. seated D. sat 对于seated的用法，首先要从动词seat说起。同学们可能只知道seat的名词用法，即只知道它表示“座位”。其实，seat还可用作动词，且是一个典型的及物动词，其意为“给某人座位”“让人坐”或“能容纳……”句式：sb be seated 或seat sb / oneself 。如： Seat the boy next to his brother. 让那个孩子坐在他哥哥旁边。 We can seat 300 in the auditorium. 我们这个礼堂可容纳300人。

高手怎么画出好看的程序流程图

高手怎么画出好看的程序流程图导语：作为一名绘图高手，自然是什么流程图都不在话下。有时候高手和菜鸟的区别并不在于有多高的技术功底，而仅仅在于一款软件的差别，用对了软件，你也可以从菜鸟瞬间转变成绘图高手。下面就让我们一起来看看绘图高手都是怎么画出好看的程序流程图吧！免费获取亿图图示软件：https://www.wendangku.net/doc/7517735380.html,/edrawmax/ 简单漂亮的程序流程图用什么软件画？想要快速制作简单漂亮的程序流程图，首选一定不是Word或者PPT的。这里推荐一款好评度非常高的流程图软件亿图图示！亿图图示（EdrawMax）是一款跨平台、多功能、同时支持云储存、分享功能的国产专业流程图绘制软件。软件内置了12000多种精美素材和实例模板，以及实时在线免费模板供用户选择。当用户完成绘制之后，可将作品导出文件为Visio、SVG、HTML、PS、JPG、PNG、PDF等多种格式。

亿图图示软件特色： 1、丰富的模板例子：亿图图示支持超过200种图表绘制，轻松绘完流程图。 2、专业的图表软件：不仅可以绘制流程图，还可以绘制组织结构图、思维导图、网络图等。 3、值得信赖的产品：超过六百万次的下载，用户遍布全世界。 4、支持流程图在线分享，生成的网页链接可以在不同的用户终端进行查看。 5、可以使用软件轻松绘制箭头、图框，让办公效率无限提升。画程序流程图的一般规则 1、用标准，使用标准的框图符号 2、按顺序，框图一般按从上到下、从左到右的方向画 3、看出入，大多数图形符号只有一个进入点和一个退出点，判断框是具有超过一个退出点的唯一符号，终端框用在开始时只有退出点，结束时只有进入点。 4、简说明，图形符号内描述的语言要简练清楚。 5、辨流向，流程线的箭头表明执行的方向，不可缺少。

软件使用手册总结

1测控系统简介本测控系统专为拉力机、压力机、电子万能材料试验机而研制。适用于测定各种材料在拉伸、压缩、弯曲、剪切、撕裂、剥离、穿刺等状态下的力学性能及有关物理参数。可做拉伸、压缩、三点抗弯、四点抗弯、剪切、撕裂、剥离、成品鞋穿刺、纸箱持压、泡棉循环压缩、弹簧拉压及各种动静态循环测试。 1.1主要功能特性 1. 硬件主控制器采用21世纪最先进的32位ARM处理器, 处理速度达到奔腾级通用计算机的水平,相比传统的8位单片机测控系统整体性能大大提高,运算速度更快,控制精度更高. 数据采集核心器件采用美国最新型超高精度24位AD，采样速率可达2000次/秒,可捕捉到力量的瞬间变化过程,全程不分档分辨力最高达500000分度。并采用独创的6点校准技术进一步提高精度，力量测量精度优于国家0.5级(最高级)标准。位移编码器计数采用4倍频技术,使位移分辨力提高4倍,最高可达0.0005mm。脉冲和电压两种输出控制方式，可控制具有脉冲或电压控制接口的任意伺服马达、变频马达或直流马达实现平滑无级调速，另还有上升、下降及停止等开关量信号输出可用于直接驱动外部继电器或电磁阀，可用于控制直流电机或气动、液压等动力装置。先进的速度、位移、力量三闭环技术，可以实现精确的任意波形控制。丰富的接口扩展能力：多达4路24位模拟量输入，3路16位模拟量输出，3路脉冲输出，3路AB相光电编码器输入，9路开关量输入，8路开关量输出，1路USB接口，1路RS232接口，1路RS485接口，4种LCD接口，1个并口微型打印机接口，1个串口微型打印机接口，1个8×4矩阵键盘接口。所有输入输出接口均采用高速光电隔离技术，具备强大的抗干扰能力。 2. 软件 Windows标准风格，层次分明的操作方式加上详尽的帮助文档和提示使之成为目前试验机行业最简单易用的软件，您的调试和软件培训效率将显著提高。采用多线程并行处理技术，测试过程中实时同时显示力量-位移、力量-时间、位移-时间、应力-应变等曲线，可随意切换到想看的曲线画面，并可查看用户设置等。标准化的测试过程控制和报表输出模版，使可以定义任意多个测试标准供用户调用，范围涵盖GB、ASTM、DIN、JIS、BS…等几乎所有测试标准。灵活强大的测试方法自定义方式,具备定速速、定位移、定力量、定力量速率、定应力、定应力速率、定应变、定应变速率等各种控制模式，可实现复杂的多步嵌套循环控制.可设置自动返回、自动判断断裂、自动归零等功能。强大的数据分析统计和曲线图形分析辅助工具，具备放大、缩小、平移、十字光标、取点等功能。多次历史测试数据可调入图形同时显示做对比分析。多达7个区间设置、40个手动取点、120个自动取点功能。具备最大值、最小值、平均值、去高低平均值、中位数、标准差、总体标准差、CPK值等多种统计功能。完全开放的测试结果编辑方法，用户可得到任何想要的测试结果。最大力、断裂力、剥离力、拉伸强度、剪切强度、撕裂强度、最大变形、屈服力、伸长率、弹性模量、环刚度、非比例延伸率、区间最小力、区间平均力、定伸长取力、定力量取伸长等多达400多个计算结果均由计算机自动算出，供用户选择调用。业界创新的Microsoft Word报表格式，简单易用，只要您会使用Word，就可编辑出您想要的精美报表。权限管理系统使您可以锁定软件的任意功能模块,将软件操作分为多个权限级别,没被授权的操作人员无法触及没被授权的模块,软件操作更加安全可靠。全数字化的校准系统，校准过程简单高效，校准数据上下位机双重保护。功能强大的单位系统，可以适应世界上任何单位制，如力值单位有gf、kgf、N、kN、tf、lbf、ozf、tf（SI）、tf （long）、tf（short）等供选择,更可扩展任意多种单位。更多重的保护机制：力量、行程、位移超量程保护设定，上下限位行程开关硬件保护设定。测试数据管理简单直观高效：单次测试数据以Windows标准的文档形式存储，自由设置储存路径和文件名。避免了传统测控软件以数据库格式储存测试数据时数据库文件会越来越大而导致软件运行越来越慢的缺点。只要您的硬盘足够大，测试数据可以无限量保存。所有操作均具有快捷键,并可连接外部手动控制盒,可外接快上、快下、中上、中下、慢上、慢下、置零、回位、测试、暂停、结束等全部常用按健. 多国语言一键切换：简体中文、繁体中文、英文，十国语言版更有日文、韩文、俄文、德语、法语、西班牙文、葡萄牙文等即将推出。绿色软件，无需安装，直接拷贝到计算机即可使用（需先安装串口驱动）,维护升级更加简单。

高手是如何快速制作出漂亮的流程图的

高手是如何快速制作出漂亮的流程图的导语：流程图的价值很高，能够让原本枯燥无味的纯文本，变得更加生动有型。高手通过流程图不断成长，值得大家学习和使用。那么，高手究竟是如何将流程图绘制的既美观又实用呢？如果你也想成为高手，可要好好阅读本文的教程哦。免费获取亿图图示软件：https://www.wendangku.net/doc/7517735380.html,/edrawmax/ 高手是用的什么流程图软件？高手绘制的流程图为什么效果好？当然是借用了专业的流程图软件来绘制。亿图图示，是一款专业的图形图表设计软件，它可用于绘制全系列的流程图。软件内置一万多个矢量符号，丰富的符号，专业的图形，帮助你快速有效绘图，还有大量现成的精美泳道流程图模板，让你快速绘制精美流程图，办公效率只增不减。

亿图图示软件特色： 1、丰富的背景样式：几十套背景样式供用户使用，且只需拖拽样式到画布即可替换背景。 2、无限量的图框：可以在画布中添加无限量的图框，大大满足用户的创作需求。 3、页面适应到绘画：画布可无限量之大，一键点击“页面适应到绘图”，软件即可自动剪切画布至绘图区域。 4、免费更新提醒：当软件有新的版本或模板更新时，软件可提醒用户进行更新操作。 5、软件咨询服务：在绘图过程中遇到操作难题，可在线与客服人员联系，并解决问题。看高手如何使用亿图图示快速绘制流程图第1步：打开软件，“新建”-“流程图”，然后根据自己的需求，选择绘图模板。比如选择基础流程图，双击鼠标即可打开绘图面板。值得一提的是，亿图图示里除了模板，还有对应的例子，如果是新手绘图，可以借鉴流程图例子帮助自己加深认识。

第2步：从左侧符号库里选择所需的图形符号，并拖动至画布中。并依次添加，直至完毕。

confident的详细用法总结大全

confident的详细用法总结大全你知道confident的用法吗?快来一起学习吧，下面就和大家分享，来欣赏一下吧。 confident的用法总结大全 confident的意思 adj. 确信的，深信的;有信心的，沉着的;大胆的，过分自信的;厚颜无耻的 n. 知己;心腹朋友; confident的用法用作形容词(adj.) 用作定语～+n. We need a confident leader to overcome these difficulties. 我们需要一个有信心的领导者来克服这些困难。 He noticed her confident smile.

他注意到她充满自信的微笑。用作表语 S+be+～+prep.-phrase I feel confident about the future of rock-and-roll music in China. 我对摇滚乐在中国的前景充满信心。 I am confident in him. 我对他充满信心。 He is confident in his ability to achieve success. 他坚信自己有能力取得成功。 We are confident in saying that the new record will be broken soon. 我们充满信心地说新的纪录很快会被打破。 S+be+～+that-clause I feel confident that we will win. 我确信我们将胜利。 confident的用法例句

1. He was confident the allies would make good on their pledges. 他相信盟友们会履行他们的承诺。 2. She has now changed into a happy, self-confident woman. 如今她已经变成一个快乐、自信的女人。 3. If there has to be a replay we are confident of victory. 如果重新比赛，我们有信心取得胜利。 4. Management is confident about the way business is progressing. 管理层对业务发展的态势充满信心。 5. Hes very forward and confident and chats happily to other people. 他很自以为是，喜欢和别人攀谈。 6. Police say they are confident of catching the gunman. 警方说他们有信心抓住那个持枪歹徒。 on holiday 还是on holidays

细说程序流程图的一般画法

细说程序流程图的一般画法导语：程序框图又被称之为程序框图，是进行程序设计的最基本依据，它的质量直接关系到程序设计的质量。绘制流程图最方便的当然还是软件啦，下面就来看看软件绘制程序流程图的一些简单画法。免费获取亿图图示软件：https://www.wendangku.net/doc/7517735380.html,/edrawmax/ 简单漂亮的程序流程图用什么软件画？想要快速制作简单漂亮的程序流程图，首选一定不是Word或者PPT的。这里推荐一款好评度非常高的流程图软件亿图图示！亿图图示（EdrawMax）是一款跨平台、多功能、同时支持云储存、分享功能的国产专业流程图绘制软件。软件内置了12000多种精美素材和实例模板，以及实时在线免费模板供用户选择。当用户完成绘制之后，可将作品导出文件为Visio、SVG、HTML、PS、JPG、PNG、PDF等多种格式。

漂亮的图片流程图是怎么画出来的

漂亮的图片流程图是怎么画出来的导语：流程图可以用于表达产品的进行过程，通过它，可以将文字可视化。一张张的图片读起来比文字要通俗易懂得多。那么那些漂亮的图片流程图都是怎么画出来的呢？一起来了解下吧。免费获取亿图图示软件：https://www.wendangku.net/doc/7517735380.html,/edrawmax/ 零基础用什么软件画出漂亮有创意的流程图？许多初学者觉得自己绘制的流程图没有别人的漂亮，不知道别人那些漂亮又有创意的流程图是怎么画出来的。要画得和他们一样其实很简单，只是你没有选对软件。如果你还在用PPT、Word绘制，那么画得不好看也是正常的。画流程图可以试试专业的流程图软件，比如亿图图示，这是一款国人设计的专业的综合性绘图软件，符合国人的操作习惯，拖拽式操作，小白也能轻松上手。软件内置海量剪贴画素材及专业符号，还有大量的实例模板，精美的配色与设计，想要不漂亮都难。它还可以一键分享到微信、微博、Facebook等社交平台供好友直接网页打开欣赏，也可以导出JPG、PNG、PDF、office、Visio等多种格式进行保存。

亿图图示软件特色： 1、来自全球超过600万的用户选择下载安装。 2、支持多系统操作：亿图图示工作流程图图可以在Windows，Mac 和 Linux上进行制作。 3、产品升级：亿图软件不断更新升级，重视用户体验度。 4、简单操作：一键式绘制工具帮助用户绘制快捷，方便使用者管理工作项目。零基础如何使用亿图图示绘制流程图 1、首先我们需要在电脑上下载安装好亿图图示软件，打开浏览器，搜索“亿图图示”，找到带有官网标识的网站，然后点击进入。

2、接着进入之后找到亿图图示的下载页面，找到对应自己电脑系统版本的下载链接进行下载，这里有三个选项，分别对应的是Windows、Mac、Linux系统。 3、下载安装好之后，打开亿图图示软件，点击“新建”---“流程图”，这个时候可以看到右侧有很多的流程图模板可以使用，我们可以使用这些模板来快速创建，也可以点击右侧的“创建”来新建一个空白模板。

干货site的使用方法总结

语法格式： site : 网址关键词或者关键词site : 网址注意事项： 1、site:后边跟的冒号必须是英文的“:”，中文的全角冒号“：”无用 2、url前不能带http:// 3、url后边不能带斜杠“/”，其实是哪里都不能带/ 4、url中不要用www，除非你有特别目的，用www会导致错过网站内的内容，因为很多网站的频道是没有www的,也就是二级域名。其他说明： 1、关键词既可以在“site:”前，也可以在“site:”后，搜索结果是一样的，但是不管谁前谁后，关键词和“site:”之间必须空一格。 2、对于“site:”搜索，关键词一样可以是多个，多个关键词之间以空格隔开。 3、支持与其他复杂搜索语法混用，各语法和关键词之间空一格 4、除了网站，还可以搜索网站的频道，但仅限于不用“/”的。 5、一个网站可能有多种语言，所以选择“搜索所有网站”和“搜索中文(简体)网页”是有差别的当然，如果指定的网站只有一种语言，怎么选择就都一样了用途： 1、可用于限制网站类型，学术资料在edu、org中会更精练，政府相关的在gov中也许更容易找。 2、用了edu、org、net、gov之类的域名后缀，并不会搜索所有含这个后缀的网站。只会搜索以这个后缀结尾的网站，带cn、us、si等各国家和地区域名后缀的edu.jp、https://www.wendangku.net/doc/7517735380.html,、org.it 等是不搜的，所以你要另外搜 3、搜索某种语言或某个关键词在指定国家的网站。 4、有的网站没有提供站内搜索，或者它的信息结构混乱，内容又多，不好找东西，那么可以用“site:”对这个网站进行检索。 google的“site:”功能比多数网站自己的站内检索还要好用，如果你查的不是动态数据库，而且对时效性要求不高的话。 5、搜索不欢迎你搜索和免费使用的网站、数据库的部分内容。 6、用“site:”搜索死链接网站、已关闭网站内的信息。

常见一般流程图怎么画

常见一般流程图怎么画导语：在日常生活中，随处可见很多的流程图，我们可以通过绘制流程图，来清晰的了解自己每一件事的流程以及步骤。接下来小编就来告诉大家如何使用软件来绘制一般的流程图。免费获取亿图图示软件：https://www.wendangku.net/doc/7517735380.html,/edrawmax/ 专业的工作流程图软件推荐亿图图示是一款跨平台、多功能、同时支持云储存、分享功能的国产专业流程图绘制软件。软件提供矢量基础的20000个以上的符号、1000个以上的模板和例子以便用户绘图使用。同时，亿图图示也支持多种类型文件的导入导出，导入如Visio、SVG，导出如Visio、SVG、HTML、PS、JPG、PNG、PDF、Office文件等等。

亿图图示软件特色： 1、丰富的模板例子：亿图图示支持超过200种图表绘制，轻松绘完流程图。 2、专业的图表软件：不仅可以绘制流程图，还可以绘制组织结构图、思维导图、网络图等。 3、值得信赖的产品：超过六百万次的下载，用户遍布全世界。 4、支持流程图在线分享，生成的网页链接可以在不同的用户终端进行查看。 5、可以使用软件轻松绘制箭头、图框，让办公效率无限提升。亿图图示绘制工作流程图优势亿图图示软件里内置大量的图标符号，可供使用者直接使用。工作流程图符号主要可分为：工作流部门符号、工作流步骤符号和工作流对象符号。所有的符号均为矢量图标，方便使用者进行拉伸、填充等操作。另外，丰富齐全的符号，将大大节省使用者的绘图时间。基本的绘图符号工作流部门符号：工作流是一种对工作流程及其各操作步骤之间业务规则的抽象、概括描述。工作流程部门主要指的是流程图的主体。

漂亮的流程图怎样画

流程图是用来表达某一信息的载体，流程图也算是图表的一种展现格式，所以不管是在绘制中还是使用中技巧都是比较多的，现在有很多绘制流程图的软件，但是其原理都差不多，在之前我们会用Word，Excel来绘制流程图，但是渐渐的发现很多功能在其中都无法实现，这话死后就要选择专业绘制流程图的软件机芯绘制使用，俗话说术业有专攻，下面和大家分享漂亮的流程图怎样画的操作技巧。绘制流程图有什么注意事项？流程图的整体框架是由流程图图形搭建而成的，我们要对流程图图形的代表含义进行了解掌握，这样才能绘制出更加完美的流程图。流程图怎样画？【寻找适合工具】小编是工具派，所以绘制流程图时需要借助工具辅助进行，首先是对工具进行选择，选择合适的工具如虎添翼，在百度搜索引擎中搜索在线编辑流程图网站，在展示的结果中选择合适的工具进行使用。

【新建流程图】选择完成之后进入网站中，这里要绘制的是流程图，所以选择流程图进行新建就可以。

【熟悉绘制面板】新建流程图之后进入流程图制作页面，在面板中汇集流程图图形，工具栏，以及流程图名称相关操作这样在使用时可以很容易的找到。【流程图框架搭建】准备工作完成之后就要对流程图进行绘制了，面板左侧的流程图图形中选择搭建框架，但是要切记流程图图形代表的含义，这样绘制的更加精美。还有连接线也要进行搭建。

【文本填充】双击流程图图形可以对里面的文本内容进行输入，在输入的同时右侧会有工具栏可以对文本的字体样式以及排列方式进行整改。

【丰富流程图】在右侧工具栏中还有样式选项，里面可以对流程图的背景颜色进行设置，有渐变色可以选择，并且还可以设置间距，透明度等操作。【导出流程图】绘制完成的流程图可以保存或者导出进行存储存使用，文件选项中可以实现此操作。不同的使用场合需要的格式也不相同。

SiteMaster的使用方法

一、测试仪表预调选择测试天线的频率范围（1）按ON/OFF按钮打开SiteMaster。（2）按MODE键。测量模式选择频率-驻波比域,然后按F1 软键。输入天线系统的下限（“Lower”）频率MHz值，按ENTER 键。（3）再按F2 软键。输入天线系统的上限（“Higher”）频率MHz值，按ENTER键。在显示区域显示新的频率数值范围FREQ scale。检查是否与输入的频率范围一致。二、测试仪表较准（1）将测试口扩充电缆（the test port extension cable）连到测试端口。若在扩充电缆端口校准，则测出的天馈线长度以此点为参考点。若在标准测试端口校准，则测出的天馈线长度以点为参考点。（2）接校准器，按START CAL 键，开始校准。三、输入天馈线的参数（1）按MODE 键。选择故障定位-驻波比菜单。（2）按DTF帮助软键。（3）按电缆损耗(LOSS)菜单。输入要测试的天馈线类型的每米的损耗dB值(7/8硬馈线，型号为LDF5-50A，cable loss=0.043dbm/m；1/2的软跳线，型号为LDF4-50A，cable loss=0.077dbm/m) ，然后按ENTER.

注意: 只有采用供货商提供的正确值，才能保证测试结果的可靠性。（4）再按传播速率PROP V 菜单。输入relative velocity (7/8硬馈线，型号为LDF5-50A，Vf=0.89； 1/2的软跳线，型号为LDF4-50A，Vf=0.88)，然后按ENTER键。注意：也可调出电缆表，直接选中所用的电缆型号。若有几种电缆混用，则选择使用最长的电缆型号。可省略（3）（4）两步。（5）按ENTER键返回主菜单。

流程图的画法,各图形详解

流程图的画法 2009年10月26日星期一 13:33 1、各司其职的形状在我的流程图中，适用于不同目的和功能的形状都有各自确定的规范。到目前为止，我一共定义了以下一些形状：（1）开始和结束作为整张流程图的头和尾，必须标清楚到底具体指哪个页面，以免日后出现歧义。（2）网页如你所见，网页的形状是一个带有漂亮的淡蓝色过渡效果的长方形，它的边框为深蓝色，中间写明了这个网页的用途，括号中的数字代表这个形状所对应的demo 文件的名称（比如这里是2.html），我有时会把流程图输出为网页的形式，并把每个网页形状和它所对应的demo文件链接起来，这样查看起来非常方便。对OmniGraffle来说这是小菜一碟，如果你被迫用Visio，嗯…… 另外，所有从形状出来的线条，都具有和此形状边框一样的颜色。这样的做法不仅看起来漂亮，在复杂的流程图中还能轻易地标明各形状的关系。我没有见过类

似的做法，所以这是由我首创也说不定，呵。（3）后台判断很常见的一个形状。我在用法上有一点和其他人的不同在于，我几乎总是让 ‘是’的分支往下流动，让‘否’的分支向右流动。因为流程图一般都是从上向下、从左到右绘制的，遵循上述规则一方面可以让绘制者不用为选择方向操心，另一方面也方便了读者阅读。（4）表单错误页既然有表单，当然会有错误信息。其实这个信息很重要，用户出错时惶恐不安，就靠着错误提示来解决问题了。你不在流程图里说什么时候显示错误页、不在demo里提供错误页，有些程序员会直接在网页上写个“错误，请检查”，所以UI设计师一定要对这个东西重视起来。但一般来说也没必要把每种错误都在流程图中表示出来，因为含有两个文本框的表单就有三种出错情况了，多了就更不用说了。所以我都是把错误页变为表单的附属页，比如表单页的编号为2，那么此表单错误页的编号就从2.1开始排下去，每种错误放到一个附属页中，这样程序员在拿到demo时也能搞清楚什么意思。结合网页和表单的形状，一个表单验证的流程图就是这样的：

怎样使用WPS文字快速制作美观的流程图

怎样使用WPS文字快速制作美观的流程图来源：互联网作者：佚名时间：10-09 16:40:46【大中小】点评：流程图简明易懂，在工作中应用很广，经常出现在文档中，下面我们就来探讨一下怎么样用WPS 快速高效和画出漂亮的流程图。流程图简明易懂，在工作中应用很广，经常出现在文档中，下面我们就来探讨一下怎么样用WPS快速高效和画出漂亮的流程图。我们利用WPS给我们提供的如下工具来画出美观规范的流程图： 1.自选图形—流程图图形 2.自选图形—连接符 3.绘图网格工具 4.对齐与分布工具 5.其它美化工具个人觉得，画好流程图的要点在于统一各框型的风格，注意它们的大小，协调它们的摆放位置。在开始之前，建议大家打开绘图网格(绘图工具栏—绘图—绘图网格)，如图中所示，选上“对象与网格对齐”，选上“在屏幕上显示网格线”，并且将“垂直间隔”选上以显示垂直网格线，根据笔者个人经验，A4纸使用0.5CM的水平间距和0.5CM的垂直间距比较合适。图1 首先画出图形框，可以一边画一边添加文字，也可以先画出整体构架，再一个个添加文字。使用了网格，要画出等大小的框就容易多了(当然也可以直接复制出等大小的方框)，稍微注意一下各方框中间的距离，注意一下长宽，就能画得比较美观，如下图所示：

图2 要选中多个图形时，使用绘图工具栏上的“选择对象”工具比较方便。注意：如果不使用网格，也务必要使用“对齐与分布”工具。要做出美观的图表,最重细节，切记，做好了图表中的每一个细节别人可能不会有赞言，但若是其中有哪个细节未处理好，是一定有损整体的形象的，会给别人留下不好、不专业的印象的。这点再怎么强调也不为过。如果有图形在画完之后发现不对，又要改成其它的图形，选中该图形，在自选图形窗格里单击要更改为的图形，点击下面的“更改自选图形”按钮即可快速更改。右键单击方框，选择“添加”文字，按CTRL+E或者点击工具栏的居中对齐按钮，使文字居中。在上图中，有三个方框，其中第一个方框中有两行字，但是文本框的下边框遮住了一部分文字，这时，在该文本框内部的文字里单击右键，选择“段落”，打开“段落”对话框，将“文本框”选项卡里的内部边距值进行调整。上图中的第二个文本框就是前者在将上、下、左、右边距都设为了0之后的结果。图3 在画好框型，添加好文字之后，可调整一下各图形之间的距离，改善整体效果。有这样几个原则一定要注意：

个人学法用法情况总结

个人学法用法情况总结我镇20××年普法依法治理工作在镇党委统一领导下,认真践行"三个代表"重要思想,围绕社会稳定、经济发展开展各项工作,为构建平安和谐先市营造了良好的法制环境。现将今年以来我的学法用法情况报告如下: 一、加强领导,健全制度,确保依法治镇工作顺利进行。一是调整充实了真法建办成员,完善了矛盾调处中心建设,制定和健全了矛盾纠纷排岔调处制、党委中心组学习学法制度、干部职工学习学法制度、普法依法治理工作宣传教育和普法依法治理工作责任制度等。二是年初根据工作实际,及时制?1?7?1?7了《法制宣传教育和普法依法治理工作意见》,10 月份又研究制定了《全民法制宣传教育的第五个五年规划》,使学法用法工作有安排,有措施,做到了有条不紊地进行。三是落实责任,明确目标。年初与各村(社区)各单位签订目标责任书落实责任,政府机关各办所及职能部门制定出依法行政、公正执法、争创平安办所计划,并通过验收合格。二、认真总结经验教训,切实开展形势多样的宣传活动。一是推广"四五"普法工作中全民学法的经验,召开现场推广会,把后坝、庙高村的经验予以推广。http:///3221788 (使用请双击此处删除页眉文字) 专业好文档为您倾心整理,谢谢使用二是及时表彰在普法依法治理工作涌现的先进单位和个人,有4 个村(社区) 单位和6 个个人获得表彰。三是充分利用三月法制宣

传月、"6.26"禁毒日、"12.4"法治宣传日、流行性传染病防治等重要日期开展多种形式的法制宣传教育。据不完全统计,宣传活动中散发宣传资料2 万多份,上街设点咨询4 次,接受群众咨询80 多次,有力地宣传了法律法规和党在农村的各项政策。四是认真搞好青少年学生的法制教育,落实了师资、教材、课时、计划,并组织法制副校长、关工委负责人等到校上法制课4 次,使3000 多学生普遍接受到了法制林剑教育。五是组织开展了"一日助贫"和向受灾户送暖和活动,机关职工、村社区干部、单位职工等140 多人次参与,捐款捐物折合人民币8000 多元。三、不断推进依法治理工作。政府继续聘请常年法律顾问,做好规范性文件清理。坚持按季度公开政务、财?1?7?1?7、村务,将依法治村(社区)、治企、校列入工作议事日程。认真开展打击两抢一盗、禁毒、非法生产烟花爆竹、乙脑防治、森林火灾防治等专项治理活动。加强部门之间的配合协作,清理整顿教育、卫生、城建、网吧等违法经营行为。按时排查调处各类矛盾纠纷,维护社会稳定。全年共排查调处纠纷公文写作首选网站-- 公文网74 件,成功71 件,调处率100,成功率96。四、努力做好信息收集上报工作。http:///3221788 (使用请双击此处删除页眉文字) 专业好文档为您倾心整理,谢谢使用鼓励干部职工群众积极收集上报信息,综治办、司法所等写出"四