英文题名：
Construction of amiRNA Binary Vector for Candidate Sterile Gene in PTGMS Line HD9802S and Genetic Transformation
中文关键词：
水稻;;HD9802S;;ami RNA双元表达载体;;遗传转化;;Southern blot
中文摘要：
光温敏核不育水稻是一种重要的种质资源,目前已知光温敏核不育基因分布广泛,在不同的光温敏核不育系中差异较大。籼型温敏核不育系HD9802S的育性受一对主效隐性基因控制,被定位于水稻第二号连锁群上的SSR分子标记RM5897和RMAN8之间的46.5kb范围内,该基因暂命名为HDtmS。
围绕该不育基因,本研究做了以下4个方面的工作：①在BGI Rise Genome Database数据库中检索位于该区间的序列(编号为Scaffold002359),并通过Fgenesh软件对Scaffold002359序列预测了3个基因共7个ORF,利用在线设靶软件共选择了21个靶位点。②以miR528天然结构为基础,利用一步PCR反扩法将21个靶位点替换miR528的茎序列,而侧翼及环结构不变,从而获得含有目的表达单元的供体载体(pDONOR-HDtms),再通过MAGIC方法将供体载体与受体载体(p1301-gfp)接合,从而获得amiRNA (Artificial microRNA)双元表达载体。③通过遗传转化,获得再生植株。④利用PCR技术检测组培苗,将阳性植株移栽至大田,通过Southern blot检测田间转基因植株的拷贝数,观察部分植株的花粉育性并统计结实率。
现已获得7个amiRNA载体的转基因阳性植株,分别为p528-3、p528-6、 p528-8、p528-10、p528-15、p528-18、p528-19。其中以上靶位点分别获得转基因植株8、4、6、6、5、5、4株。PCR及Southern blot检测表明目的表达单元已成功导入籼稻9311中；而花粉育性检测试验表明：p528-8阳性植株花粉大都不育,结实率也很低。
英文摘要：
The Photoperiod and Thermo-sensitive Genic Male Sterile Lines (PTGMS) rice is an important germplasm resource. The PTGMS gene distributes extensively, expresses differently in variety of PTGMS line. The fertility of Indica TGMS line HD9802S was controlled by one pair of recessive genes and named HDtms temporarily. Previous studies identified HDtms on chromosome2as the locus between the SSR molecular marker RM5897and RMAN8, which contained46.5kb DNA fragments.
In this study we concentrate on the following four aspects.
1. The46.5kb DNA fragment (No.Scaffold002359) was found in the database of BGI Rise Genome Database, then three unknown genes with7ORF were forecasted in fragment No.Scaffold002359by using Fgenesh software, with which we designed21amiRNA targets.
2. Based on the structure of miR528, we constructed an amiRNA vector. In order to accomplish this process, we firstly changed the structure of donor vector (pDONOR-HDtms) by replacing a21bp length sequence on miR528's stem with the fragment of our target sequence. Then we combined the donor vector (pDONOR-HDtms) with host vector (p1301-gfp) by MAGIC (Mating-assisted genetically integrated cloning).
3. We transfered the amiRNA vector into agrobacterium EHA105, and infested cultivated-plants using genetic transformation.
4. Positive plants were chosen by PCR, then transplanted to field. The copy numbers of the positive plants were tested by southern blot. Lastly, we observed pollen fertility and analyzed seed setting rates of these transgenic positive plants.
We constructed seven amiRNA vectors with comparable genetic modified plants, and the vectors were p528-3, p528-6, p528-8, p528-10, p528-15, p528-18, p528-19, respectively. PCR and Southern blot analysis indicate that the target genes have been successfully transferred into indicia9311. The result of field test shows that p528-8-positive plant is male sterility with low seed setting rate.
