利用单片段代换系图位克隆水稻种子休眠性新基因Sd7-1

Seed dormancy is a important trait associated with pre-harvest sprouting (PHS) and the seed storage quality. A new gene controlling the seed dormancy,Sd7-1,was identified by using of the single segment substitution line(SSSL) and was delimited to a physical region of 800 kb. In the study,Sd7-1 gene will be farther delimited to a phycical gegion of 30kb and annotated by using of a larger mapping population with more than 3000 individuals.Primers will be designsed according to the candidate ORFs and used to identify the target ORF by sequencing and RT-PCR. The expression time and position of the target ORF will be examined using RT-PCR or Real time-PCR. Subsequently, the full-length DNA of target gene will be obtained by using of LA-PCR, and the homologous genes and functional information of Sd7-1 in different plant species will be analysed by comparative sequence analysis. The function of the target gene will be confirmed by using of funtional complementary test of Agrobacterium-mediated transformation and RNA interference. Genetic effect will be analysed using SSSLs with Sd7-1 from different donors, and alleles will be identified at molecular level. The clone and allelic identification of Sd7-1 gene will helps to elucidate the genetic basis of rice seed dormancy at molecular level and lay solid basis on molecular breeding of rice seed dormancy.

水稻种子休眠性是影响穗发芽和种子贮藏质量的重要性状。本项目组利用SSSL鉴定出一个控制水稻种子休眠性的新基因Sd7-1并将其界定在约800kb的物理区域内。本研究拟进一步扩大作图群体(>3000株)，将Sd7-1基因界定在大约30kb的物理距离内并进行基因注释；利用RT-PCR和Real time-PCR技术对候选ORF进行表达分析并测序，确定目的ORF及其表达情况；扩增目标区段、测序比对，获得目的基因的全长DNA序列，并分析Sd7-1基因在不同物种间的同源基因及功能信息；采用农杆菌介导转化法进行转基因功能互补试验和采用RNAi干涉技术对目的基因进行功能验证；筛选携带Sd7-1基因而供体来源不同的SSSL进行基因效应分析并从分子水平上鉴定其等位变异。Sd7-1的克隆及等位基因鉴定将有助于从分子水平上进一步阐明水稻种子休眠特性的遗传基础，为水稻抗穗芽的分子设计育种奠定基础。

水稻种子休眠性是影响穗发芽和种子贮藏质量的重要性状。本项目组利用SSSL对水稻种子休眠性基因Sd7-1进行了精细定位，将其定位在约48.9 kb 的物理范围内，该区域存在9个候选基因。根据9个候选基因在W22s 和 W0 之间的序列比对和荧光定量表达分析，初步判定候选基因ORF6为目标候选基因，该基因全长4622 bp，其中CDS长度为750 bp，含有4个外显子，共编码250个氨基酸，编码一个F-box结构域的蛋白；荧光实时定量PCR结果表明ORF6在抽穗后20d的种子中表达量最高且表达差异显著；构建了ORF6基因敲除载体pC1300-Cas9 –LCBg1-g2、过表达载体pC 1300-Act-ORF6-OE和互补载体pC1300-ORF6，并通过农杆菌介导法转化到W0中，获得了基因敲除转基因苗；发现Sd7-1基因在不同材料间存在3种等位基因类型；研究了W0和W22s种子发育过程中内源激素的变化及其对外源激素GA3和ABA的响应。Sd7-1基因的克隆及等位基因鉴定将有助于从分子水平上进一步阐明水稻种子休眠特性的遗传基础，为水稻抗穗芽的分子设计育种奠定基础。