| Rice(Oryza sativaL.) is one of the most important crops in the world and become a model plant for many reasons:the relatively small genome size; the genome co-linearity with other monocots; the mature transformation techniques. As an effective strategy to study gene function, T-DNA insertional mutagenesis has been widely adopted for constructing mutant libraries. In this study, we have screened, identified and analyzed of the library of rice T-DNA inserted mutants which for ZH11 as receptor.This library included 62 independent transgenic lines. In all these transgenic lines, we have got 50 lines which had transgenic marker(bar) among which 16 lines had single copy T-DNA tag and 4 lines (late flower mutants T744,T745,T754 and early senescence(T761) were the mutants which caused by T-DNA insertion. In the same time, we have gene mapped the early senescence mutant T761.1. Sixty-two independent transgenic lines mutated form Zhll have been investigated and screened. The traits include heading date, plant height, leaf color, plant type, flower development and so on. In this library,24 percents of these mutants are heading date mutants,40 percents are the plant height mutants,8 percents are the leaf color mutants,3 percents are the plant type mutants and 12 percent are the tiller number mutants. On the other hand, we have constructed the gene mapping groups used early senescence mutant. After all of these, we can confirmed that T744,T745,T754 and T761 (early senescence) were caused by single copy T-DNA insertion.2.Used Inverse PCR, TAIL-PCR, PCR walking, Anchor PCR and Developed Anchor PCR to amplify of T-DNA flanking sequence in T745, T754 and T761.We have got the backbone sequences of vector in T754 and non-specific sequences in T745.3. We have gene mapped the early senescence mutant (T761), which turn up rust staining on the leaves in seeding stage, then the apex of leaves become scorch 5-7 days after the leaves came out and after the leaves become full length, the top third of the leaves die. Besides these, its heading date is delayed and its ripening rate is much lower.To study the genetic laws of T761 (early senescence mutant), we have hybrided it with 9311,G46B, HuanB, Japonica and SH527 etal. The normal and early senescence would segregate in all the F2 populations and the segregation ratios fit to 3:1, which mean that the trait was controlled by a single recessive gene.4. SSR markers were used to gene map the T761 (early senescence). By using bulked segregation analysis on an F2 population developed from the cross of T761 and 9311. This gene have been mapped close to two SSR markers:RM480 and RM3170 on chromosome 5, with genetic distance of 4.5cM and 1.1 cM. We named this gene ess1(t) provisionally.On the other hand, we have backcrossed T761 with ZH11, in order to find if the mutant was caused by the T-DNA insertion. So we have amplified the bar gene in all the 102 early senescence rice from F2 population by PCR, and have got 98 positive individualplants. We can say that the mutant is due to the insertion of T-DNA. |
