Site-Directed Mutagenesis Of Sh2Gene And Function Analysis

Maize is a kind of cereal crop which is grown the most widely and yielded thegreatest. Maize occupies a very important position in economic production, it is notonly an important food crop but also the main industrial raw materials, such as starch,pharmaceutical, oil, molasses, alcohol industry. Genetic improvement to cereal cropshas aroused scholars’ wide concern, and conventional breeding combination withtransgenic breeding can effectively speed up the breeding process.Maize genome size is about2,500Mb, contains about50,000genes whichco-regulated with yield, quality, resistance and other traits. How to isolate and clonecandidate genes related to each good trait from the large bank of genes has become animportant issue in transgenic breeding.Sh2gene encodes a regulatory subunit of maize endosperm AGPase, which isclosely linked to the activity of AGPase. As the key enzyme of starch anabolic, highlyexpression of AGPase can effectively improve the starch content and the overall yieldof maize. Currently, the relevant genetic transformation of wild-type Sh2gene inmaize has been studied extensively, but research on mutant Sh2genes was rarelyreported.In this study, we isolated wild-type Sh2gene from the endosperm of maize inbredH99, and successfully cloned mutant gene HS33/Rev6Sh2by the method ofsite-directed mutagenesis.To further study the function of HS33/Rev6Sh2gene, pCAMBIA3301plasmid andthe CaMV35S promoter are used in this experiment, and successfully constructedplant expression vector.Wild-type Arabidopsis (Columbia) is infected by the method of "floral dip", and weobtained the T2transgenic plants after the detection of PCR and RT-PCR. We foundthat the bolting period of transgenic Arabidopsis is postponed in varying degreescompared with controls, while the mature seed number in per silique increased by 13.0%and34.6%respectively under the growth condition E1and E2. In addition,compared with the controls, the1000-seed weight of transgenic Arabidopsisdecreased under E1, while increased by13.8%under E2.To obtain excellent maize germplasm of high-starch and high-yield, HS33/Rev6Sh2gene was transferred into maize immature embryos and somatic embryosmediated by Agrobacterium tumefaciens. At present, we have obtained six positive T0generation plants of HS33/Rev6Sh2gene which were confirmed by PCR.