Study On Cloning Of Rice (Oryza Sativa L.) Starch Synthesize Key Enzyme Gene And Molecular Regulation

Starch is the major component of yield in the world's main crop plant and the predominant reserve carbohydrate.Starch plays a key role in human activities as a source of nutrition for humans and animals,and as a feedstock for industry.Based on the recent advances in the biochemistry and molecular biology study of starch biosynthesis enzymes,manipulation of starch quality by gene engineering is in progress in order to understand the molecular events in starch synthesis and to improve starch quantity and quality.In this research,the protein coding region(coding sequence region,CDS region) of rice granule-bound starch synthaseâ… (GBSSâ… ,WAXY) and rice starch branching enzyme 3(RBE3) have been isolated and purified from rice.For isolate the genes, designed specific oligonucletide primers based on the sequence of starch biosynthesis enzyme genes in GeneBank,then used the way of the polymerase chain reaction(PCR) and the reverse transcriptase-PCR(RT-PCR) to enriching the gene.Isolated genes include full-length of Waxy and RBE3 from rice cDNA,as well as their fragments from rice genomic DNA.The full-length CDS region of rice Waxy gene was isolated from rice (Nipponbare) immature seed cDNA.The rice Waxy CDS is 1,830 bp,encoding a polypeptide of 609 amino acids.There are two bases difference when compared the rice Waxy CDS to the known sequence,producing two amino acids mutation,but the activity of protein wasn't changed.The sequence of Waxy fragment isolated from rice genomic DNA,and the fragment of length 206 bp was the same as the known sequence.The full-length CDS region of rice RBE3 gene was isolated from rice (Nipponbare) immature seed cDNA.The rice Waxy CDS is 2,478 bp,encoding a polypeptide of 825 amino acids.There are two bases difference when compared the rice RBE3 CDS to the known sequence,producing one amino acid mutation,but the activity of protein wasn't changed.The sequence of RBE3 fragment isolated from rice genomic DNA,and the fragment of length 195 bp was the same as the known sequence.The plant expression vectors suitable for rice transformation were constructed with the rice Waxy and RBE3 gene in overexpression,RNAi or multiple gene expression(one gene is overexpressed,the other is restrained) under the control of promoter GluA-3 originated from rice glutelin 3 or HMW promoter from wheat glutenin.Mediated by Agrobacterium,the fusion gene was introduced into rice varieties Zhonghua 11.Transgenic plants carrying the RBE3 gene in RNAi were obtained and verified by PCR and Southern blot.By semi-quantative RT-PCR showed that the expression of RBE3 was significantly reduced in transgenic immature seeds. Analysed the amylase content in endosperm of these transgenic plants,the amylose content was increased.The average increase level was about 140%,while the increase level for some individuals was significant and came to 238%.In all transgenic lines, the thousand-seed weight was reduced.The average reduction level was about 47%.