红麻中与atp9嵌合且共转录基因发掘及其转录本加工方式分析

Chimeric genes were key genes associated with cytoplasmic male sterility (CMS), they all existed specifically in the CMS lines. Our previous studies showed that a gene combined and co-transcripted with atp9 was found in kenaf, the chimeric gene in its region was associated with CMS, and the chimeric gene existed in the CMS line, its maintainer and F1 progeny, it was different from that of other plants. But the sequence structure and the post-transcriptional processing model of the chimeric gene were unknown. Given all this, the RNAs were isolated from the CMS line, its maintainer and F1 progeny ( bred by Ruiyang Zhou) of kenaf. Then the key chimeric gene associated with cytoplasmic male sterility in kenaf was developed using Northern blot methods, and whose sequences structure were further analyzed. Then the post-transcriptional processing model of transcripts co-transcripted with atp9 were further analyzed based on CR-RT-PCR methods. These results revealed that the key cutting of DNAs at defined sites led to two phenotypes of sterile and fertile based on different modification of RNA posttranscription. These results could provide direct and important evidence for illuminating the molecular mechanism on the CMS of kenaf, Meanwhile they could accumulate theoretical basis for revealing the mechanism on the CMS of other plants.

嵌合基因是引起作物细胞质雄性不育（CMS）的关键基因且特异存在不育系中。我们的前期研究表明，红麻中存在与线粒体atp9基因嵌合且共转录的嵌合基因，该嵌合基因所在区域与红麻CMS相关且存在模式不同于其它作物，不仅存在不育系而且存在保持系和F1代中，但该嵌合基因的序列结构以及转录本的加工方式尚不清楚。鉴于此，本项目以周瑞阳等选育的红麻细胞质雄性不育系、保持系及F1 (不育系/恢复系) 代为材料，采用Northern blot和环化RNA反转录PCR（CR-RT-PCR）方法，找出与红麻atp9嵌合且共转录的嵌合基因以及该嵌合基因转录本的修饰切割位点，分析嵌合基因的结构特点及其转录本的加工方式，以期探明红麻嵌合基因转录本的不同加工方式分别导致不育、可育两种表现型的关键修饰切割位点。本项目的研究结果不仅为揭示红麻CMS分子机理提供重要的直接证据，而且也为其它作物CMS分子机理的阐明积累基础资料。

红麻细胞质雄性不育（CMS）系的选育与杂种优势利用已取得较大进展，但红麻CMS的分子机理仍未阐明。以红麻细胞质雄性不育系UG93A、保持系UG93B以及F1代为材料，主要获得以下结果：（1）atp9基因未与nad5基因片段嵌合。基于红麻线粒体基因组数据，明确了atp9基因的3’端确实存在nad5基因片段，但未发现囊括atp9基因和nad5片段在内的开放阅读框即嵌合基因。（2）明确了atp9完整mRNA和成熟mRNA转录起始和终止位点。 UG93A、UG93B和F1中atp9的完整mRNA 5’端转录起始位点分别位于起始密码子上游-691bp、-863bp、-691bp处，3’端转录终止位点分别位于起始密码子下游+1141bp、+2106bp、+1141bp处；UG93A，UG93B和F1中的 atp9 的成熟mRNA 5’端转录起始位点均位于起始密码子上游-71bp处，3’端转录终止位点分别位于起始密码子下游+535bp、+661bp、+661bp处。不育系UG93A转录本比UG93B和F1少126bp即转录提前终止。明确了atp9基因的转录特征为5’端只有一个转录起始位点，而3’端存在多个转录终止位点，有别于atp1，atp4，atp6和cox3的5’端存在多个转录起始位点，但3’端仅存在1个终止位点的转录特征。（3）明确了atp9基因转录本差异与不育或可育的关系，转录本发生剪切，不育系转录本短于保持系和F1。具有相同不育细胞质的UG93A和F1的完整mRNA转录起始和终止位点相同，但与UG93B不同，经剪切和加工成成熟的mRNA时，具有可育性状的保持系和F1具有相同的转录本，而具有不育性状的不育系转录本短于保持系和F1。. 本项目从RNA水平全面阐明了atp9转录本的修饰加工方式导致不育、可育两种表现型的关键修饰切割位点，为揭示红麻CMS分子机理提供重要的直接证据。