野生稻成熟胚愈伤组织褐化相关基因克隆及功能分析

Callus browning and Low Differentiation frequency are the main factors restricting the genetic transformation of indica rice varieties, reducing callus browning rate and increasing differentiation frequency are important ways to improve the efficiency of indica rice genetic transformation. Using an introgression line population derived from Yuanjiang common wild rice (Oryza rufipogon Griff.) and the elite O. sativa indica cultivar Teqing, we cultured mature embryos, screened and acquired the introgression line YIL25 with low callus browning rate. With the F2 population from YIL25 backcrossed with Teqing, we initially positioned the QTL（qB3-2）on chromosome 3 which controls callus browning trait. Through identifying genotype for a total of 4800 plants from F2、F3 and F4 and selecting exchange individuals to identify callus-browning trait, we mapped qB3-2 within the 24.8kb region, and identified the candidate gene Cb-1 combining with sequence and expression analysis. Basing on this, in this project we want to clone the Cb-1 gene from Yuanjiang common wild rice, verify its function, explore it maybe involving in signal pathways, identify culturability and genotype of rice main variety resources and evaluate the possibility of Cb-1 as a transgenic safe selection marker, which will not only provide a theoretical basis for elucidating mechanism of rice callus browning but also lay an important foundation for the establishment of high efficient safe indica rice variety genetic transformation.

愈伤组织易褐化、分化频率低是籼稻遗传转化的主要限制因素，降低愈伤组织褐化率、提高分化频率，是提高籼稻转化效率重要途径。我们利用元江野生稻与特青构建的渗入系群体，通过成熟胚培养，筛选出愈伤组织褐化率低的渗入系YIL25，利用YIL25与特青回交的F2群体，在第3染色体检测到控制愈伤组织褐化性状QTL（qB3-2），通过对总计4800个F2，F3，F4单株进行基因型分析，选择交换单株鉴定愈伤组织褐化性状，将qB3-2定位在24.8kb的区间，结合序列分析和表达分析，确定了候选基因Cb-1。本项目将在此基础上，克隆来自元江普通野生稻的Cb-1基因，解析其生物学功能，鉴定水稻主干品种资源的组织培养力及基因型，评估Cb-1作为转基因安全筛选标记的可能性。研究结果不仅为阐明水稻愈伤组织褐化的分子机理提供参考，而且为建立高效安全的籼稻品种遗传转化平台提供奠定重要基础。

籼稻愈伤组织易褐化，严重影响籼稻遗传转化。野生稻作为栽培稻的祖先种，含有许多栽培稻中丢失的等位基因，本研究利用以愈伤组织褐化较重的籼稻特青为背景的褐化较轻的元江野生稻渗入系YIL25，对降低愈伤组织褐化性状进行精细定位及基因克隆，分离出降低愈伤组织褐化基因BOC1，BOC1编码一个SIMILAR TO RADICAL-INDUCED CELL DEATH ONE (SRO)蛋白，BOC1适量表达降低愈伤组织褐化，进而提高遗传转化效率。BOC1可能通过抑制氧化胁迫引起的细胞衰老和死亡来降低愈伤组织褐化。BOC1启动子区Tourist MITE转座元件特异存在于部分野生稻中，可提高BOC1在愈伤组织中表达。因此，野生稻中BOC1等位基因可用来改善栽培稻组织培养特性和遗传转化效率，提高水稻分子育种水平。另外，我们筛选出37份愈伤组织褐化较轻的水稻材料，其中包括8份野生稻和29份栽培稻，为水稻分子育种提供良好的遗传转化受体。