水稻白叶枯菌抗性相关基因R1×4选择性剪接机制及功能初探

【作者】 裴雁曦；

【导师】 李德葆；

【作者基本信息】 浙江大学 ， 植物病理与分子生物学， 2002， 博士

【摘要】 本论文前期工作中，通过mRNA差别显示获得了三个受白叶枯菌(Xanthomonas oryzae pv.oyzae)诱导表达的差异cDNA片段B1、B2、B3。经分析推测是同一基因的不同剪接方式。进一步实验证实三个片段表达受到白叶枯菌诱导。国际互连网BLAST查询表明该基因为新基因。本课题旨在对这一与白叶枯菌抗性相关且存在多种转录形式的基因进行进一步研究，了解其不同转录形式的特征，表达特点、多转录本间的相互关系及功能等。 通过cDNA末端快速扩增(RACE)获得了该基因完整cDNA序列(命名为RIX4)，确定了正确的开放阅读框架：应用M-EST芯片技术得到了RIX4基因另一转录本RIX4-4I片段，以RT-PCR的方法扩增并克隆了该转录本的全长序列；基于3’RACE技术，使用多重引物扩增，获得大小不同的多种产物(转录本)，进一步的克隆分析正在进行中；从水稻IR26DNA中克隆了RIX4基因2661bp的DNA序列。 对所获得的转录本进行了序列分析表明：RIX4基因包含有7个外显子和6个内含子，除第四内含子外，全部具备植物内含子极端保守的／GT-AG／特征：B1、B2、B3三个转录本是RIX4基因在转录过程中前端两个选择性剪接组件发生了选择性剪接的产物，剪接组件首尾具有CAAG碱基特征；B1是三个转录本中最长的一个，开放阅读框(ORF)1530bp，完整包含有该基因所有7个外显子部分；B2、B3的ORF分别为306bp和222bp；RIX4-41是RIX4基因保留了第四内含子的一个全长转录本，ORF共1626碱基。至此共分离克隆了RIX4基因的四个全长转录本和该基因的DNA序列。 互联网生物信息学分析和预测表明，RIX4是一种不稳定的水溶性蛋白，等电点4.6左右，定位于核内；它具有N端糖基化位点、蛋白激酶C磷酸化位点、酪蛋白激酶磷酸化位点、十四烷酰化位点、核定位信号、酰胺化位点、低复杂性位点、Pfam-B2301区域等，在130-165氨基酸之间还存在有四次重复的“VQDEMNAQP”氨基酸序列；其蛋白质的二级结构由a螺旋(Alpha helix)、延伸带(Extended strand)、随机卷曲(Random coil)三种结构模块组成。RIX4-4I与RIX4所预测结构基本相同。RIX4基因氨基酸序列互联网数据库搜索，仅发现与拟南芥RAD21类蛋白(染色体粘合蛋白)有一定的同源性，且具有该类基因尾部较为保守的Pfam-B2301模体特征，加之定位于核内，推测RIX4基因属于水稻中这一基因家族成员，可能在有丝分裂或减数分裂中起作用，不正常表达可能会影响水稻育性。 通过RFLP多态性分析，将RIX4基因定位于水稻第8号染色体 构建了RIX4基因正义与反义植物表达载体，通过农杆菌法对水稻粳稻品种中花11进行转化，获得正义表达转化株41株，反义22株。对各株(系)PCR验证表明阳性率约94％。正、反义转化株系的种子结实率差异极显著。进一步验证了该基因属于染色体粘合蛋白基因家族成员的可能。 应用水稻2200条非冗余表达序列标签所制备的cDNA微阵列及同位素”33P杂交体系，对水稻抗白叶枯菌近等基因系中早14R、9S经白叶枯病菌诱导后特异基因表达模式、信号传导、 浙江大学博士学位论文2 RIX4基因的表达特点进行研究。结果表明：表达丰度上调基因315个，下调176个。其中 78个受诱导的己知功能基因。cDNA ＃阵列上表达变化基因反应了植物防卫反应中基因表达 的协同作用基本模式：白叶枯菌侵染下乙烯介导的信号传导途径在防卫反应中可能起重要作 用；试验结果还提供了一些未明确与植物抗病有直接关系，但在供试材料受白叶枯菌诱导后 表达量发生了明显改变的基因以及多个受白叶枯菌强烈诱导但功能未知的基因。RIX4基因 在两系中受诱导后表达丰度轻微上调。更多还原

【Abstract】 Three differential expressed cDNA fragments （B1, B2, B3） were obtained by DD-PCR in rice callus mRNA of IR26 （resistance to Xanthomonas oryzae pv. oyzae; Xoo） and jingang30 （susceptible to Xoo） in previous work.. Sequence identity among them were 70-80%. It was confirmed by Northern that their expression were induced by Xoo. BLAST searching showed they are novel genes. It was proposed that the mentioned three cDNA fragments might be different splicing pattern of a same gene, R1X4.This work was designed to study further the splicing pattern of this gene, the relationship among them, expression characteristics and the function.3’ RACE and 5’ RACE were carried out Result showed that the ORF of RIX4 gene has 1530 bp, and the kozak frame confirm the ORF. Another transcripts, RIX4-4I was cloned by M-EST microarray. Based on 3’ RACE, some other transcripts were obtained and sequence analysis were on studing. RIX4 DNA were cloned from rice IR26. Expression of RIX4 gene was analysed using RT-PCR. Expression abundance in IR26 was similar to that in jingang30. But in IR26, the expression was induced by Xoo mightily. RIX4 gene might be related to rice resistance to Xoo.The sequence of RDC4 gene and the transcripts were analysed. Result showed that this gene has six introns and seven exons, and was 266Ibp long. All the intron has the /GT-AG/ consensus characteristics except for the forth intron. Bl, B2, B3 were the different transcripts of the same gene produced by alternative splicing. ORF of the transcript Bl has 1530bp that had all the six introns of this gene excised; B2 was the same with Bl but the first alternative splicing component was cleavaged, and with ORF of 306 bp; B3 was the tanscript has all the two alternative splicing component cleavaged, and with ORF of 222bp. CAAG at me splicing site were the same characteristic of these two alternative splicing components. Transcripts RIX4-4I had five of the six intron excised, and retained the forth intron which has % base pair.Bioinfomatics analysis showed that R1X4 was a soluable protein, pI4.6, localized in nucleus. There were N-glycosylation site, Protein kinase C phosphorylation site, Casein kinase II phosphorylation site, N-myristoylation site, Amidation site, Tyrosine sulfation site, Bipartite nuclear location site, low complexity, Pfam-B₂301 and "VQDEMNAQP" four times repeats in mis protein.Search in the internet database showed that R4 protein was partial identity with Arabidopsis RAD21 family and has the conservative Pfam-B₂301 domain in the end. In addition, RDC4 was localized in nucleu. So, It was proposed that RIX4 gene was one of the RAD21 gene family in rice and play a important role in mitosis and meiosis.Sense and antisense plant expression vector were constructed. Through agrobacteriwn hanefaciens-mediated transformation of japonica rice varieties Zhonghuall, 41 （sense） and 22 （antisense） transgenic plants were obtained, respectively. It was proved that 94% of theregenerated plants showed positive by PCR. The setting percentage of the sense and antisense transgenic plants were different distinctly.RIX4 was mapped on chromosomes 8.The expression profiles, signal transduction, RIX4 gene expression were studied using cDNA microarrary containing 2200 Expression Sequence Tags （ESTs）. The leaves of rice near isogenic line （NIL） zhongzaoHR （Resistance） and 9S （Susceptible） were induced with Xanthomeneus oryzae pv. oyzae （Xoo） for 3 hours at 4-leaves stage .315 genes were up-regulated while 176 genes were down-regulated by the induction. Further studies on the 78 induced genes with known functions indicated that the changes of gene expression pattern not only reflect coordinated gene regulation in plant defense but also show the basic model of plant in response to pathogen. Novel genes induced by Xoo were also found. Further studies indicated that ethylene signal pathway may play important role in rice defense response while infected by Xoo. The expression of RIX4 was upregulated slightly in this experiment.更多还原