【作者】 张胜利；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2007， 博士

【摘要】 分蘖现象是水稻、小麦等禾本科作物在生长发育过程中的一种特殊的分枝特性,它直接决定水稻、小麦等的穗数及其产量,而且与创建理想株型密切相关。稻属中广泛分布在热带、亚热带地区的野生稻具有丰富的遗传多样性,以及许多优异的栽培稻中已灭绝了的基因,是栽培稻产量、品质、抗性以及其它农艺性状改良的重要的基因库。随着粳稻日本晴(Oryza sativa L. ssp. Japonica cv. Nipponbare)和籼稻93-11(Oryza sativa L. ssp. Indica cv. 93-11)基因组计划的完成及测序成本的日趋下降,开展核苷酸水平的比较基因组学研究变得越来越现实。因此,为有效的利用稻属野生资源提供一定理论和技术支撑,本研究在序列水平采用比较基因组学的方法,并结合生物信息学技术,对整个稻属各基因组类型植物中的MONOCULM1(MOC1)直向同源区进行了比较分析,得出如下重要结论。比较基因组学方法是一个提高基因注释准确性的强有力的手段。通过RT-PCR试验及随后测序方法发现了NCBI对粳稻日本晴的一个基因注释有误,NCBI的注释多出了一段长15个核苷酸的序列。稻属中多数直向同源基因是很保守的,且多外显子基因的外显子-内含子结构也很保守,基因种类及其排列顺序也都表现出了较高的保守性。此外,还发现了一些个别基因组特有的基因。在本研究所有的BAC中存在直向同源基因的是MOC1和探针42DP1006所来自的基因。其它的基因只是在一些BAC中有直向同源基因,而另一些BAC中则没有,这主要是由于某些基因组类型的野生稻BAC较小,序列不够长所致,同时某些MOC1同源BAC中(尤其是四倍体)有较多的重复序列导致基因密度很低,也是该同源区所共有的直向同源基因少的重要原因。稻属中各MOC1同源区平均每个基因的外显子数目相差不大。AA组基因密度较高,四倍体基因组中(除CCDD基因组外)两个MOC1同源BAC均表现出一个基因密度较高,而另一个基因密度很低的现象。发现了一例整个基因序列完全从头形成的新基因产生过程。通过序列比对发现,从稻属植物中亲缘关系较远的GG基因组,开始只有一段长度有限的保守的DNA序列,到CC组保守序列逐渐变长,到BB组和AA组就形成了一个新基因,即日本晴MOC1 PAC上的第13号基因及其在AA组和BB组中的直向同源基因,该基因有全长cDNA(AK062635)支持(100%)。在多个稻属物种中找到了基因组加倍或基因加倍后复制基因去功能化的分子证据。稻属四倍体物种中除CCDD基因组的O. alta外,探针42DP1006的两个同源基因中均有一个在多次重复RT-PCR试验中均未扩增出任何产物。GG基因组的O. granulata中,该同源基因发生了串联复制,形成前后两个同源拷贝并被逆转座子插入而分开,其中上游的拷贝在多次重复RT-PCR试验中也均未扩增出任何产物。因此,推测加倍后的复制基因发生了去功能化。发现了一个在基因密度、基因表达上比较特殊的四倍体O. alta。在本研究所涉及的MOC1同源区内,CCDD基因组O. alta中的两套遗传物质都有较高的基因密度(CC,9.8kb/基因;DD,10.1kb/基因),且探针42DP1006的直向同源基因对都保持了一定表达能力。此外,O. alta中的日本晴MOC1 PAC上第6号基因的一个同源基因虽被长约4kb的一个逆转座子插入到第3个内含子中但并没有影响该同源基因的表达,而且该四倍体的另一个同源基因在表达上也是正常的。稻属植物基因组膨胀的主要动力是转座子和逆转座子扩增。研究发现稻属植物中基因组较小的AA组(350Mb~448Mb)和FF组(338Mb)具有较多的MITE(AA,16个~21个;FF,30个)和Tc1-IS630-Pogo(AA,18个~34个;FF,32个);而其它基因组较大(除BB组423Mb外,均在650Mb以上)的则这两种DNA型转座子较少。从LTR型逆转座子的数量上来看,情况正好相反,即AA组、FF组较少,而其它基因组类型则较多。从转座子和逆转座子累计长度占BAC序列长度的比重上看,也表现出了较大基因组类型中逆转座子所占比重较大,且转座子所占比重也不低的趋势。这暗示,稻属植物膨胀主要是由于转座子和逆转座子扩增造成的,而且有研究表明这可能是生物界基因组膨胀的普遍规律。用邻接法(Neighbor-Joining)、最大简约法(Maximum Parsimony)、最小进化法(Minimum Evolution)等构建的比较一致的MOC1基因进化树表明,稻属植物中与AA基因组亲缘关系较近的是BB组(包括四倍体中的BB组),其次是CC组(包括四倍体中的CC组),而后是DD组和EE组,HHJJ、HHKK、GG、FF等组与AA组间亲缘关系较远。更多还原

【Abstract】 Tillering is the ramifying trait emerging in the period of growth of some grass species e.g. rice, wheat, et al.. Tillering that is close correlate with the creating of ideal plant shape is the main deciding factor of spike numbers and the consequent yield of rice, wheat, et al. The wild rice species distributed in the Torrid Zone and sub-Torrid Zone possess abundant genetic diversity and excellent genes that do not exist in cultivated rice varieties. So, the wild rice species become important gene pools for improving yield, grain quality, stress resistance, and other agronomic traits of cultivated rice varieties. Along with the completion of genome project of Oryza sativa L. ssp. Japonica cv. Nipponbare and Oryza sativa L. ssp. Indica cv. 93-11 and the sequencing cost dropping more and more, to do some work about comparative genomics at DNA sequence level become more and more realizable. So, in order to give some theoretical and technological support for the wild rice resources utilizing effectively, the comparative analysis among MONOCULM1 (MOC1) orthologous regions in Oryza genus has been done by employing the methods of comparative genomics at DNA sequence level and bioinformatics. The results are depicted as follows.The comparative genomics is a strongly method to improving the veracity of gene annotation. One gene annotation error in NCBI was discovered through RT-PCR and subsequently sequencing. 15bp nucleotides in the coding sequence of this gene annotated in NCBI is excessive than the reality of this gene.The majority of orthologous genes and the intron-exon structures of these genes in Oryza genus are much conserved and the gene contents and order is also. Some genome specific genes were found. The orthologous genes existed all over in the BACs sequenced in this research are MOC1 and the gene that probe 42DP1006 is come from on Japonica MOC1 PAC. Other genes on MOC1 PAC except the two mentioned above can be found their orthologous genes from several BACs only. The reason that some orthologous genes cannot be found are mainly the two, short BAC sequences and more repeat appearing in the BACs especially in the BACs created by tetraploid.The exon number per gene on average in each MOC1 homologous BAC is similarity with each other. High gene density is discovered in AA genome type, but one high and one low are found in tetraploid genomes except CCDD genome. One whole cds sequences of new gene generated by de novo have been detected here. by sequence alignment, we find a short conserved DNA sequence comparison within far relative species GG, FF and EE, but the conserved sequence become longer than before gradually within more closely relative species EE and BB and the new gene which has full length cDNA support (AK062635) by 100 percent is originated eventually in AA and BB genomes.The molecular evidence for nonfunctionalization of duplicated gene originated from whole genome duplication and/or gene duplication has been discovered from several species of Oryza. There is one homologous gene of 42DP1006 in all tetraploid Oryza species except CCDD genome has no PCR products in RT-PCR experiments although several times repeated. The homologous gene of 42DP1006 in GG genome, O. granulata, has been duplicated in series and divided by retrotransposon insertion. The upriver homologous cannot be amplified in several times RT-PCR. So, nonfunctionalization of duplicated gene can be presumed.A tetraploid, O. alta, which has an especial gene density and expression comparing with other tetraploid in Oryza genus has been found. Both the subgenome CC and DD of O. alta have high gene density and both the orthologous gene pairs of 42DP1006 can be transcribed to some extend. Further more, the expression of orthologous gene of MOC1 PAC No.6 in O. alta is normal though the third intron of this gene is inserted by about 4kb retrotransposon. The other one of the orthologous gene pairs can be transcribed normally too.The driving force of genome expansion of Oryza genus is the amplification of transposons and retrotransposons. we find the smaller genome size of AA(350Mb~448Mb)and FF(338Mb)possess more MITE (16~21 for AA,30 for FF)and Tc1-IS630-Pogo(18 ~34 for AA,32 for FF), but not for the larger genomes. From the aspect of LTR retrotransposon number, the reverse condition appeared comparison with above, i.e. smaller LTR retrotransposons detected in AA and FF genome than other genome types in Oryza genus. From the aspect of sequence proportion by the accumulated sequence length of transposons and retrotransposons to BAC length, the larger genome size possess high proportion of retrotransposons and less high proportion of transposons. This implies the genome expansion of Oryza genus is the result of amplification of transposons and retrotransposons. and it maybe the law of genome expansion in biology.According to the consensus MOC1 phylogenetic tree constructed by Neighbor-Joining, Maximum Parsimony, and Minimum Evolution, the most close relative genome type with AA genome that contain cultivar rice is BB and then the CC, DD, EE, respectively. The genome type of HHJJ, HHKK, FF and GG have alienated relative with AA genome.更多还原