【作者】 黄华磊；

【导师】 柴友荣；

【作者基本信息】 西南大学 ， 遗传学， 2007， 硕士

【摘要】 十字花科中，拟南芥（Arabidopsis thaliana）是重要的模式植物，芸薹属含有多种重要的油料、蔬菜和观赏园艺植物。芸薹属内部的物种间以及芸薹属与拟南芥之间的比较基因组学研究，是理论研究一个热点，也是将拟南芥模式植物中功能基因的重要研究成果应用到芸薹属作物进行遗传改良的有力工具。甘蓝型油菜（Brassica napus）是由甘蓝（B．oleracea）和白菜型油菜（B．rapa）融合而成的异源四倍体。比较基因组学发现，芸薹属基本种在进化中相对于拟南芥发生了基因组水平的三倍化，并伴随发生了局部区段的丢失、再加倍和重排。目前的比较基因组学研究多集中于通过分子标记手段，从宏观上揭示物种间在基因组和染色体水平的共线性程度，而基于功能基因比较克隆的比较基因组学和比较生物学研究对于油菜等芸薹属作物具有重要的理论和现实意义。黄籽性状具有综合品质优势和良好的市场前景，是世界油菜研究中的一个热点和难点。白菜型油菜和甘蓝中均存在表型稳定的天然黄籽基因型，但甘蓝型油菜却没有，而通过远缘杂交等方法创造黄籽材料的选育周期长、效率低，尤其是黄籽表型易受环境影响而不稳定。当前急需对甘蓝型油菜和其亲本物种中与黄籽性状相关的重要功能基因进行全长序列的比较克隆，然后进行比较基因组学和比较生物学研究，揭示这3个物种间在黄籽性状上的分子遗传学机理，并通过基因工程改良甘蓝型油菜的籽色性状。拟南芥等植物中种皮色素的主要成分是原花青素单体的聚合物，是由苯丙烷-类黄酮途径形成的。该途径的众多功能基因中，拟南芥TT12基因（AtTT12）在种皮中特异表达，编码一种类多药转运蛋白，负责种皮色素向液泡的转运，其单基因失活突变即可导致由野生型的深褐色种子转变为透明种皮（黄籽）。油菜与拟南芥同属十字花科，种皮色泽等性状也相似，因此本研究克隆了甘蓝型油菜和其亲本物种中TT12基因的全长序列，进行了比较基因组学分析。1)率先克隆了甘蓝型油菜及其2个亲本物种的TT12基因（家族）本研究采用cDNA末端快速扩增技术，率先克隆了甘蓝型油菜及其亲本物种白菜型油菜和甘蓝中TT12基因（家族）的全长cDNA和基因组序列。甘蓝型油菜BnTT12-1基因全长2712bp，mRNA全长1747bp（不计poly A尾巴，下同）；BnTT12-2基因全长3000bp，mRNA全长1678bp；白菜型油菜BrTT12基因全长2711bp，mRNA全长1747bp；甘蓝BoTT12基因全长3062bp，mRNA全长1739bp。该结果为深入研究芸薹属TT12基因的功能、进化、调控模式奠定了基础，也为通过对TT12基因的转基因表达抑制（反义、RNA干扰）或TILLING突变体筛选来创造新型黄籽油菜新材料奠定了基础。2)BnTT12-1、BnTT12-2、BrTT12和BoTT12符合TT12基因的典型特征它们的基因组序列都有7个内含子，位置都与AtTT12的一致，都符合GT…AG的内含子剪切位点边界序列特征。在BnTT12-1、BnTT12-2、BrTT12和BoTT12 mRNA的62-1585、17-1540、60-1583和78-1601bp处都有一个1524bp的ORF（含终止密码子，下同），5’UTR分别为61、16、59和77bp，3’UTR分别为162、138、164和138bp。它们的poly A加尾信号AAATAAA分别位于最末poly A加尾位点上游间隔96、83、98和83bp处。与AtTT12相似，推导的BnTT12-1、BnTT12-2、BrTT12和BoTT12蛋白均为507 aa。BnTT12-1的计算分子量Mw=55.080 kDa，等电点pI=3.24；BnTT12-2的Mw=55.168 kDa，pI=3.24；BrTT12的Mw=55.199 kDa，pI=4.24；BoTT12的Mw=55.128 kDa，pI=3.24。它们是典型的酸性蛋白。它们均以亮氨酸含量最高（15.11％）。BnTT12-1、BnTT12-2和BoTT12有21个潜在的磷酸化位点，BrTT12则有19个，所以磷酸化可能参与它们的蛋白活性调节。预测它们没有信号肽或信号锚定，很可能定位于质膜，而且都有9个可能的跨膜区，这与跨膜转运蛋白AtTT12相似。它们的二级结构非常相似，均以α螺旋为主（64.69％、62.52％、64.89％、64.10％），其次是随机卷曲（20.71％、21.10％、19.72％、18.93％）。整个分子平均分布大量的α-螺旋，但其中在蛋白的中部偏N末端、偏C末端以及C末端，都有较大的α-螺旋，同时还有一些小的α-螺旋分散在整个分子中，而延伸链、β-转角和随机卷曲的分布则相对较为均匀，这与TT12等MATE家族跨膜转运蛋白的基本特征一致。未能预测出它们蛋白质的三级结构。核苷酸序列和氨基酸序列的BLAST分析、序列两两比对和多重比对、序列的系统发生分析均表明，它们与AtTT12有最高的相似性。从基因结构、蛋白结构、序列同源性三大方面分析，均显示它们是AtTT12的垂直同源基因，具有TT12的典型特征。3)芸薹属基本种中TT12基因数与拟南芥相比并没有发生三倍化本研究通过竭尽式克隆，从甘蓝型油菜、白菜型油菜和甘蓝中分别只克隆到了2条、1条和1条TT12基因。Southern blot杂交结果所指示的同源拷贝数与基因家族成员数的序列结构非常吻合。因此，芸薹族的公共祖先与拟南芥分开后在TT12位点上很可能并没发生基因数目的三倍化，或者在基因加倍后不久随即发生了多数成员的丢失，从而使芸薹属二倍体基本种中只有1条TT12基因。4)白菜型油菜和甘蓝确实为甘蓝型油菜的供体亲本BnTT12-1和BrTT12之间基因组序列的一致性达到96.5％，BnTT12-2和BoTT12之间达到99.4％，远远高于甘蓝型油菜种内成员BnTT12-1与BnTT12-2之间的一致性，氨基酸水平上的趋势也是如此。核酸水平和氨基酸水平的系统发生聚类显示，BnTT12-1和BrTT12更紧密，BnTT12-2则和BoTT12更紧密。此外，从内含子的一致性、5’UTR中的oligo A的位置与长度、基因序列中的特征性变异碱基、氨基酸序列中的特征性变异氨基酸等方面来看，都表明BnTT12-1对应于BrTT12，BnTT12-2对应于BoTT12，说明甘蓝和白菜型油菜的确为甘蓝型油菜提供了遗传物质。本研究从功能基因家族成员的全长序列比较克隆的角度，为揭示甘蓝型油菜与其亲本物种间的进化关系提供了直观而具体的分子证据。5)揭示了TT12基因的一些新的结构特征芸薹属4个TT12基因的5’UTR中均存在转录起始位点的多态性，BnTT12-1中为A₁、A₆，BnTT12-2中为A₁、A₂，BrTT12中为A₁、G₃，BoTT12中为A₁、A₅、A₃₀、A₃₅、G₅₉。它们的3’UTR中也都存在加尾位点的多态性，BnTT12-1中为C₂₆₂₇、T₂₆₃₁、C₂₆₉₄、T₂₇₁₂，BnTT12-2中为T₂₉₂₉、C₂₉₃₉、C₃₀₀₀，BrTT12中为C₂₆₂₄、C₂₆₈₈、C₂₆₉₁、T₂₇₁₁，BoTT12中为T₂₉₉₄、T₃₀₄₀、C₃₀₆₂。它们也许代表了一种顺式调控方式，也许只是转录起始和poy A加尾过程中的允许误差。ATG上游的oligo A结构是十字花科TT12基因的一个保守性基序。BnTT12-1中为22bp，与ATG间隔7bp；BnTT12-2中为10bp，与ATG间隔6bp，BrTT12中为25bp，与ATG间隔7bp，BoTT12中为10bp，与ATG间隔6bp，AtTT12中为10bp，与ATG间隔21bp。这种oligo A结构有可能在mRNA翻译时有助于核糖体的特异识别与结合，因此值得深入研究。6)构建了共抑制BnTT12、BrTT12和BoTT12（家族）的反义植物表达载体将它们共保守的589bp的反义片段构建到中间载体pCambia2301G中，替换GUS基因，由CaMV 35S启动子驱动，形成了反义共抑制所有成员的植物表达载体，命名为p2301G-TT12A，为通过基因工程抑制内源TT12基因的表达从而创造转基因新型黄籽油菜奠定了基础。更多还原

【Abstract】 In Brassicaceae, Arabidopsis thaliana is an important model plant, and Brassica contains many important oilseed, vegetable and ornamental species. Comparative genomic study both within Brassica and between Brassica and Arabidopsis is a hotspot in theoretical study, and is a powerful tool to bring the research results on functional genes achieved in Arabidopsis into effect in genetic improvement of Brassica crops. B. napus is an amphidiploid of B. oleracea and B. rapa. Comparative genomic study has revealed that the genome of diploid species of Brassica has experienced a triplication process as compared with that of Arabidopsis, accompanied by loss, reduplication or rearrangement of local regions. Mainly using molecular marker means, present comparative genomic studies mainly focus on revealing the degree Of collinearity between mega regions of genomes, while comparative genomic study and comparative biological study based on comparative cloning of functional genes has both theoretical and applicable implications for Brassica crops.With outstanding comprehensive quality advantages and prospective marketability, yellow seed trait is a hotspot, but also a nodus, of rapeseed research worldwide. Both B. oleracea and B. rapa have stable-phenotyped natural yellow seed genotypes, but B. napus has not. And creating yellow seed stocks through means like distant hybridization is time-consuming and low efficient, and the expression of the yellow seed phenotype shows drastic sensitivity to environmental factors. At present, it is highly necessary to simultaneously clone important functional genes involved in seed color determination from B. napus and its parental species, and then perform comparative genomic and comparative biological studies. This will help to clarify the molecular genetic mechanism of yellow seed trait in these 3 species, and lay the base for improvement of seed color trait of B. napus through genetic engineering.The major constitutents of seed coat pigments of plants like Arabidopsis are polymers of proanthocyanidin monomer, which is synthesized via phenylpropanoid-flavonoid pathway. Among the many functional genes of this pathway, A. thaliana TT12 （AtTT12） is specifically expressed in the seed coat, and it functions to transport seed coat pigments into the vacule by encoding a Multidrug transporter protein. Single gene mutation of AtTT12 leads to the turning of the seed color from the dark brown wild-type to transparent testa （tt, yellow seed） type. Since B. napus and Arabidopsis both belong to the same family Brassicaceae and share similar seed color trait, this research performed isolation of full-length TT12 genes from B. napus and its parental species, and carried out comparative genomic analysis of these genes.1) Cloning of TT12 genes from B. napus and its parental speciesUsing rapid amplification of cDNA ends （RACE） technology, full-length cDNAs and corresponding genomic sequences of TT12 genes from B. napus and its parental species were isolated. B. napus BnTT12-1 gene is 2712 bp with an mRNA of 1747bp （not including poly A tail）, and BnTT12-2 gene is 3000 bp with an mRNA of 1678 bp. B. rapa BrTT12 gene is 2711 bp with an mRNA of 1747 bp. B. oleracea BoTT12 gene is 3062 bp with an mRNA of 1739 bp. This result lary the base for study of function, evolution, and regulatory mode of TT12 genes of Brassica, and for creation of novel yellow seed stocks by suppression of intrinsic TT12 gene expression （via antisense or RNA interference） or by LILLING mutants screening.2) BnTT12-1, BnTT12-2, BrTT12 and Bo TT12 conform to typical features of TT12They all have 7 introns with the same positions as those of AtTT12. All the introns conform to canonical intron splicing boundary sequence "GT...AG". At 62-1585, 17-1540, 60-1583 and 78-1601 bp of BnTT12-1, BnTT12-2, BrTT12 and BoTT12 mRNAs, there are an open reading frame （ORF） of 1524 bp （including stop codon）. Their 5’ UTRs are 61, 16, 59 and 77 bp, and 162, 138, 164 and 138 bp for 3’ UTR, respectively. Their poly A tailing signal AAATAAA is located upstream the latest poly A tailing site with an interval of 96, 83, 98 and 83 bp respecitvely.Like AtTT12, the deduced BnTT12-1, BnTT12-2, BrTT12 and BoTT12 proteins all are 507 aa in length. BnTT12-1 has an Mw of 55.080 kDa and a pI of 3.24, while 55.168 kDa and 3.24 for BnTT12-2, 55.199 kDa and 4.24 for BrTT12, and 55.128 kDa and 3.24 for BoTT12, respectively. They are typical acidic proteins. Leucine is the richest （15.11%） one in their amino acid compositions. BnTT12-1, BnTT12-2 and BoTT12 have 21 potential phosphorylation sites, and BrTT12 has 19, implying that phosphorylation might be involved in regulating their protein activity. They were predicted with no signal peptide or signal anchor, while they were predicted with the higheset possibility to be located on cytoplasmic membrane since each of them possesses 9 predicted significant transmembrane domains. These features imitate the membranen-associated transporter AtTT12.The 4 proteins share very similar secondary structures,αhelix is the most abundant proportion （64.69%, 62.52%, 64.89% and 64.10%）, followed by random coil （20.71%, 21.10%, 19.72% and 18.93%）. The whole molecule is dominated by a large number ofαhelices, especially largeαhelices exist at near-N-terminus, near-C-terminus and C-terminus regions. Extended strands,β-turns and random coils distribute nearly evenly along the whole protein. These features is similar to those of TT12-type MATE family transmembrane domain proteins. Their tertiary structure could not be predicted currently.BLAST at both nucleotide and amino acid levels, pairwise-and multi-alignment of sequences, and phylogenetic anylysis all indicate that these 4 genes share the highest homologies with AtTT12. Clues from gene structure, protein structure and sequence identities all suggest that BnTT12-1, BnTT12-2, BrTT12 and BoTT12 are orthologous genes of AtTT12.3) In Brassica basic （diploid） species, the TT12 gene was not triplicated as compared with AtTT12Through exhaustive cloning, this research only isolated 2, 1 and 1 TT12 gene（s） from B. napus, B. rapa and B. oleracea respectively. Southem blot results coincided with this result. Hence, it is concluded that, after divergence with Arabidopsis, the TT12 gene in Brassiceae has not been triplicated, or most of the triplicated members were lost right after the triplication event, leading to the single-copy situation in current Brassica diploid species.4) B. rapa and B. oleracea both really are parental species of B. napusBnTT12-1 and BrTT12 share 96.5% of identities on whole genomic sequence scale, and 99.4% between BnTT12-2 and BoTT12. These homologies are much higher than that between BnTT12-1 and BnTT12-2, which are intra-species paralogs. Ananlysis with amino acid sequences also gave the same trends. On phylogenetic trees of both nucleotide and amino acid sequences, BnTT12-1 groups with BrTT12 first, and BnTT12-2 groups with BoTT12 first. Furhermore, clues from intron similarities, position and length of the oligo A structure in the 5’ UTR, and featured mutation sites on both nucleotide and amino acid sequences, all point to the corresponding relationships of BnTT12-1 to BrTT12 and BnTT12-2 to BoTT12, suggesting that B. rapa and B. oleracea are donors of genetic substances of B. napus. This research provided straight and concrete evidence for revealing the evolutionary relationships among B. napus, B. rapa and B. oleracea, based on a profile of comparative cloning of full-length functional gene family.5) Some new structural features of TT12 are revealedThe 4 Brassica TT12 genes all have alternative transcription iniation sites in the 5’ UTR, BnTT12-1 at A₁ and A₆, BnTT12-2 at A₁ and A₂, BrTT12 at A₁ and G₃, and BoTT12 at A₁, A₅, A₃₀, A₃₅ and G₅₉, respectively. In their 3’ UTRs, altenative poly A tailing sites were detected, BnTT12-1 at C₂₆₂₇, T₂₆₉₄, C₂₆₉₄ and T₂₇₁₂, BnTT12-2 at T₂₉₂₉, C₂₉₃₉ and C₃₀₀₀, BrTT12 at C₂₆₂₄, C₂₆₈₈, C₂₆₉₁ and T₂₇₁₁, and BoTT12 at T₂₉₉₄, T₃₀₄₀ and C₃₀₆₂, respectively. They perhaps represent one kind of cis-regulation, or just allowable deviations in transcription initiation and poly A tailing processes.Oligo A stretch just upstream the start codon ATG is a conserved common feature of Brassicaceae TT12 genes. In BnTT12-1, it is 22 bp, with 7 bp of interval upstream ATG, while 10 bp / 6 bp for BnTT12-2, 25 bp / 7 bp for BrTT12, and 10 bp / 6 bp for BoTT12, respectively. Interestingly, AtTT12 also has such a structure, which is 10 bp and 21 bp upstream ATG. This oligo A structure might play a role in recognition or binding of the ribosome in translation. So it deserves further study.6) Construction of an antisense plant expression vector for suppression of BnTT12 family, BrTT12 and Bo TT12 A 589-bp antisense fragment （drived by CaMV 35S promoter） conserved in BnTT12 family, BrTT12 and BoTT12 was integrated into intermediate vector pCambia2301G to replace the GUS gene, then an antisense plant expression vector p2301G-TT12A was constructed for transgenic suppression of all intrinsic BnTT12, BrTT12 and BoTT12 transcripts.更多还原