Starch Accumulation And Activities Of Key Enzymes Involved In Starch Synthesis In The Grains Of Maize Inbred Lines With Different Starch Contents And Sbe CDNA Clonging And Expressing

Maize starch, the principal constituent of maize (Zea mays L.) grain,is the oneof best starch, its purity being 99.5%, and 80% starch of all world are from maize.Maize starch has wide application prospect in food, medication, chymistry industry,weave, paper making, and so on. Maize is changing gradually from the food suppliesand feedingstuff to raw material of industry and agriculture. Nowadays, we needmaize breed with not only its high yield but also its high quality. With the developingof the world economy, the dramaticly increasing of the population and a great dealexpending of the energy sources, it is being more and more important and urgency toiucubrate the mechanism of starch biosythesis. It is very significant for selecting highyield and high quality maize breeds through biological technology to understand indetailed the mechanism of starch biosynthesis. To explain the profound mechanism,the two high-starch and tow low-starch content maize inbreds have been used asexperiment materials, the first objective of this study was to analyse the dynamicaccumulation of starch, amylose and amylopectin and to determine the patterns of theactivities of key enzymes involved in starch synthesis and the relationship to theaccumulation speed of starch, amylose and amylopectin during grain filling period.The second objective was to optimize the method of extraction SGAPs (StarchGranule-Associated Protein), and to compare the expression of SGAPs betweenhigh-starch and low-starch maize inbreds. The third objective was to clone the cDNAof sbe1 and sbe2b, and then expression the protein of SBEⅠand SBEⅡb in E.coil.Theexpected results would help to understand the mechanism of starch biosynthesis in high and low starch maize inbred lines and to provide the theoretical and practicalguidance in maize breeding and cultivating for high-yielding and good quality.The main results shows:1. Endosperm amylose, amylopectin and starch content (the sum of amylose andamylopectin content) during grain filling of the 4 inbred lines increased in "S"curve fashion until reaching a stationary phase of maximal content. Amylosecontent of 2 high-starch inbred lines were significantly higher than 2 low-starchinbred lines on 40 DAP. Amylose accumulation rate of 4 inbred lines were notsignificantly different in early grain filling stage. Amylopectin and starch contentof 2 high-starch inbred lines were constant higher than 2 low-starch inbred linesduring grain filling. Amylopectin and starch accumulation rate of 4 inbred lineswere highest on 25—30DAP during grain filling period.2. Changes in activities of ADPGPPase, SSS and GBSS of 4 inbred lines grainsappeared a single peak curves, and reaching the peak at 20—30DAP. Whencomparison was made among inbred lines, the two high-starch inbred lines hadhigher peaks of SSS and GBSS activities than the two low-starch inbred lines atearly grain filling stage. Changes in activities of SBE in high-starch inbred linegrains appeared single-peaked curves and the peak values appeared on 20 DAP,while 2 low-starch inbred lines showed a double-peak curve, the peak value of 2low-starch inbred lines appeared on 20—30DAP and 30 DAP respectively. TheADPGPPase activities of 315062-1 was the highest in 4 inbred lines at middlegrain filling stage.3. According to the correlation analyse, amylose, amylopectin and starchaccumulation rate of 4 maize inbred lines was significantly and positivelycorrelated with activities of ADPGPPase, SSS and GBSS, while grain filling ratehad no correlation with the activities of SBE except 314168-2. The results ofcorrelation analysis indicated that there were significant correlation betweenactivities of ADPGPPase, SSS and GBSS in 4 inbred lines, however Q-enzymeactivities of low-starch inbred lines were not significantly related to other enzymicactivities. The result indicated ADPGPPase, SSS and GBSS were the key enzymes contributing to the synthesis of maize starch. We speculated that theseenzymes proteins may have a coordinating action in starch biosynthesis within theamyloplast, operating as a functional multiprotein complexes.4. The finally protein cntent of maize grain depends on the rising speed of later stageof grain filling and has little relationship to the content of the beginning stage.More higher protein content at the middle stage of grain filling resulted lowerstarch content at the last, the protein cntent of later stage of grain filling has littlerelationship to the finally starch content. The protein content of grain had anegative correlation to the starch content, amylase content and amylopectincontent during the grain filling period.5. SDS is very important for extracting SGAPs from starch granulas.Our experimentresults showed that the SBEⅡb, SSⅠ,GBSSI, zein proteins relative contents ofthe sample extracted by different SDS content were different. These proteinrelative contents of the sample by 10% SDS was most, and that the SGAPs totalquality also was is the most, so the 10% SDS was at best for extracting the SGAPs.At the mention of time of sample heated up at 100℃, the SGAP total quality wasthe most when the sample had boiled for 5min, however the target proteinsrelative contents is not the most; The SBEⅡb, SSⅠ,GBSSI, zein proteins relativecontents were the most when the sample had boiled for 2—3 min but the SGAPtotal quality was not the most. Which showed SGAP total quality was more andmore with the time of ferv. Prolonging, that was because zein protein quality wasincreased, and then the target proteins relative contents is not increased,especially the GBSSI protein ralative content was the most at 2—3min.6. As to the dynamic expression modle of the SGAPs for high-and low-starch maizeinbred lines,the SGAPs total qulity was higher at middle filling period than that atearly and later filling period,and reaching a stationary phase of maximal content at20—30DAP. The expression models of the GBSSI proteins of high-andlow-starch maize inbred were similar, however the expression models of theSBEⅡb, SSⅠ, zein proteins were notable difference between high-andlow-starch maize inbred. 7. According to the sequences of sbe1and sbe2b cDNA, we desighed a couple ofprimers with restriction enzyme site corresponding to the pET32a expressionvector, and two target gene of fragments of the inbred line 415084-2 could begained by RT-PCR with their length of 2400 and 2470 respective. In comparisonwith the reported she1 and sbe2b cDNA in GenBank, nuleotide sequenceshomology of the clone sbe1and sbe2b cDNA were both 99%, and we predicted thesecondary structure and tertiary structure of SBEⅠand SBEⅡb protein byPredictProtein and ExPASy Proteomics Server, the predicting results showedSBEⅠand SBEⅡb were both sphericity protein, and that their tertiary structureswere similar.8. The sbe1 and sbe2b cDNA were inserted pET32a vector by restrictionendonuclease digestion, electrophoresis, gel reclamation, DNA Ligation and so onmolecular biological technologies, and that which were transformed to E.coilBL21(DE3). At the same time she1 and sbe2b cDNA were induced expression inE.eoil BL21(DE3) by IPTG. The results indicated that molecular weight of thefusion proteins were both about 90kD, which were the same sizes as anticioated,and confirmed SBEⅡb protein expression quantity was the most with 0.5 mmol/LIPTG.