甘蓝型油菜及其亲本物种BAN基因家族的克隆与比较基因组学分析
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摘要
甘蓝型油菜(Brassica napus)是我国最重要的油料作物之一，其黄籽类型与黑籽类型相比有着许多品质优势，黄籽性状是国内外油菜育种的热点之一。虽然作为甘蓝型油菜的亲本物种白菜型油菜(B．rapa)和甘蓝(Brassica oleracea)都有天然的表现稳定的黄籽类型，但它没有，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。
甘蓝型油菜黄籽性状形成的分子机理目前还不清楚，但可以借鉴同属于十字花科的拟南芥透明种皮(TRANSPARENT TESTA，TT)性状研究的成果。花青素苷和缩合单宁的合成共用的是一种底物，而由BANYULS(BAN)基因编码的花青素还原酶(Anthocyanidin reductase，ANR)正是在类黄酮物质合成途径中决定这种底物是否转向缩合单宁合成的关键酶。BAN将花青素还原为2，3-顺式-黄酮-3-醇的功能，随后2，3-顺式-黄酮-3-醇聚合成为缩合单宁。研究甘蓝型油菜及其2个亲本物种的BAN基因有助于进一步研究甘蓝型油菜黄籽性状形成的分子机制，为通过遗传工程创造稳定的甘蓝型黄籽油菜新材料提供基础。
本研究克隆了甘蓝型油菜BAN基因家族(BnBAN)的3个成员、白菜型油菜BAN基因家族(BrBAN)的2个成员和甘蓝BAN基因家族的2个成员(BoBAN)的cDNA序列及其基因组全长序列，并进行了系统分析，主要结果如下：
1)率先克隆了甘蓝型油菜及其2个亲本物种的BAN基因家族
本研究利用RACE技术，率先获得了甘蓝型油菜及其亲本物种白菜型油菜和甘蓝的BAN基因家族部分成员全长eDNA和对应的基因组序列。BnBAN-1、BnBAN-2和BnBAN-3分别为1571 bp、1612 bp和1574 bp，推导的mRNA分别为1178 bp、1124 bp和1180 bp(不计poly(A)尾巴，下同)；BrBAN-1和BrBAN-2分别为1607 bp和1623 bp，推导的mRNA分别为1212 bp和1174 bp；BoBAN-1和BoBAN-2分别为1543 bp和1642 bp，推导的mRNA分别为1172 bp和1150 bp。这是在芸薹属植物中首次克隆全长BAN基因，为深入研究芸薹属植物BAN基因的功能、进化、调控模式奠定了基础。
2)芸薹属BAN基因具有BAN的典型特征
芸薹属7个BAN基因都有5个内含子和6个外显子，符合GT……AG的内含子边界序列特征，并且和AtBAN的位置相符。在BnBAN-1、BnBAN-2、BnBAN-3、BrBAN-1、BrBAN-2、BoBAN-1和BoBAN-2 mRNA的41-1069、50-1066、41-1078、74-1111、46-1062、47-1063、41-1069 bp区段有分别为1029、1017、1038、1038、1017、1017、1017 bp的开放阅读框(ORF，包括终止密码子)，5’UTR分别为40、49、40、73、45、46、40 bp，3’UTR分别为109、58、102、101、112、87、103 bp。分别在它们polyA加尾位点上游的22-42、23-43、22-42、20-39、20-42、15-34、22-45 bp有可能的非典型polyA加尾信号TATAAA和AAATAAT。
BnBAN-1、BnBAN-2、BnBAN-3、BrBAN-1、BrBAN-2、BoBAN-1和BoBAN-2分别编码由342、338、345、345、338、342、338个氨基酸组成的多肽链，理论分子量(Mw)分别为37.850、37.680、38.277、38.266、37.614、37.927和37.554，等电点(pI)分别为6.38、4.93、6.38、6.06、5.00、5.81和4.87。它们都编码酸性蛋白，氨基酸组成中丝氨酸的含量均为最高。BnBAN-1、BnBAN-3和BrBAN-1有24个潜在的磷酸化位点，BnBAN-2、BrBAN-2和BoBAN-2都有18个，BoBAN-1有23个。预测它们均没有信号肽和跨膜域，可能象AtBAN一样定位于细胞质中。
芸薹属3个物种的7个BAN蛋白与AtBAN和苜蓿BAN一样，也具有典型的罗斯曼折叠，表现在都具有一个保守的富含谷氨酸的序列GXXGXXA，在该序列中它们均有同已报道的拟南芥中决定BAN底物特异性的氨基酸N_(21)和L_(22)对应的残基，同时在这7个蛋白均存在一个与AtBAN高度相似的亮氨酸拉链。它们的二级结构非常相似，以α螺旋所占比例最高，占氨基酸总数的39.77～42.60％，随机卷曲占30.04～32.84％，有少量的延伸链和β转角。它们蛋白的中部都有2个大的α螺旋，C末端也有1个。它们的三级结构也很相似，与葡萄黄烷酮醇还原酶的晶体整体相似，与赭色掷孢酵母的NADPH依赖型羟基还原酶部分相似。
从基因结构、蛋白结构、序列同源性三大方面分析，均显示芸薹属3个物种的7个BAN基因是AtBAN的垂直同源基因，具有BAN的典型特征。
3)甘蓝、白菜型油菜为甘蓝型油菜提供了遗传物质
BnBAN-1和BoBAN-1之间基因组序列和编码区序列的一致性分别高达97.5％和99.1％。BnBAN-2和BoBAN-2之间基因组序列和编码区序列的一致性分别高达96.5％和99.5％。这些种间基因的一致性高于甘蓝型油菜种内成员之间的一致性。无论核酸水平还是氨基酸水平的系统发生学聚类结果，内含子的相似度，基因序列和氨基酸序列上的特异位点均显示，BnBAN-1和BoBAN-1紧密地聚在一起，BnBAN-2和BoBAN-2紧密地聚在一起。可以确定BnBAN-1和BnBAN-2来自于甘蓝(BoBAN-1和BoBAN-2)。甘蓝和白菜型油菜的确为甘蓝型油菜提供了遗传物质。本研究从编码关键酶的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。
4)甘蓝型油菜、白菜型油菜和甘蓝BAN基因家族的成员数
采用甘蓝型油菜典型黑籽系5B的基因组DNA为材料，分别用EcoRI、EcoRV、SphI、SacI、XbaI这5种限制性内切酶进行酶切和Southern blot杂交，结果显示BnBAN家族可能存在4个以上成员。由于这3个物种BAN基因家族全部成员克隆工作尚未完成，且没有对白菜型油菜和甘蓝的BAN基因进行Southern杂交，因此有待于进一步的进行竭尽式的克隆，才能回答芸薹属二倍体基因种相对于拟南芥在BAN功能位点究竟是否符合典型的“三倍化”模型。
Oilseed rape (Brassica napus) is one of the most important oil crops. Its yellow-seeded type shows many quality advantages, and yellow seed trait is a hotspot of rapeseed breeding goal worldwide. Both parental speices, B. oeracea and B. rapa, have stable-phenotyped natural yellow seed genotypes, but B. napus has not. Yellow seed trait introgression through 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 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 molecular mechanism of yellow seed trait formation is not clear still, but achievements on the TRANSPARENT TESTA (TT) trait in Arabidopsis thaliana can be referred The anthocyanins and the condensed tannin use common substrate. The BANYULS (BAN) gene coding anthocyanidin reductases (ANR) determines whether the substrate enters the condensed tannin synthesis pathway. BAN reducecs the anthocyanidin to 2,3-cis-flavan-3-ols which are then polymerized later to form condensed tannin. Study on BAN genes from B. napus and its parental species will help to reveal the molecular mechanism of yellow seed trait formation and lay the base for transgenetic creation of stably inherited yellow seed stocks.
In this research, the full-length cDNAs and corresponding genomic sequences of 3 members of B. napus BAN gene family (BnBAN), 2 members of B. rapa BAN gene family (BrBAN) and 2 members of B. oeracea BAN gene family (BoBAN) were isolated and systemically analysized. The main results are as follows:
1) Cloning of BAN genes from B. napus and its parental species
Using rapid amplification of cDNA ends (RACE) technology, full-length cDNAs and corresponding genomic sequences of BAN genes from B. napus and its parental species were isolated BnBAN-1, BnBAN-2 and BnBAN-3 are 1571,1612 and 1574 bp, with the corresponding mRNAs of 1178,1124 and 1180 bp (not including poly A tail), respectively. BrBAN-1 and BrBAN-2 are 1607 and 1623 bp, with the corresponding mRNAs of 1212 and 1174 bp, respectively. BoBAN-1 and BoBAN-2 are 1543 and 1642 bp, with the corresponding mRNAs of 1172 and 1150 bp, respectively. This is first report of full-length BAN gene cloning from Brassica species, and will lay the base for study of function, evolution, regulatory mode of BAN genes of Brassica.
2) BnBAN,BrBAN and BoBAN conform to typical features of BAN
They all have 5 introns with the same positions as those of AtBAN. All the introns conform to canonical intron splicing boundary fearture sequence "GT...AG". At 41-1069, 50-1066, 41-1078, 74-1111,46-1062,47-1063 and 41-1069 bp of BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 mRNAs have an open reading frame (ORF) of 1029,1017,1038,1038,1017,1017 and 1017bp (including stop codon). Their 5' UTRs are 40,49,40,73,45,46 and 40 bp, and 109,58,102,101,112,87 and 103 bp for 3' UTRs, respectively. Their possible non-canonical poly A tailing signals TATAAA and AAATAAT are located 22-42,2343,22-42,20-39,20-42,15-34 and 22-45 bp respectively upstream the poly A tailing sites.
The deduced BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 proteins are 342,338,345,345,338,342 and 338 aa in length, with Mw of 37.850,37.680,38.277,38.266,37.614,37.927 and 37.554 kDa, and pI of 6.38,4.93,6.38,6.06,5.00,5.81 and 4.87, respectively. They all are typical acidic proteins. Serine is the richest one in their amino acid compositions. BnBAN-1, BnBAN-3 and BrBAN-1 have 24 potential phosphorylation sites; BnBAN-2, BrBAN-2 and BoBAN-2 have 18; and BoBAN-1 has 23. They were predicted with no signal peptide and transmembrane domain, with the higheset possibility to be located in cytoplasm.
Like AtBAN and MtBAN, the 7 Brassica BAN proteins have classical Rossmann fold characterized by a conserved glycine-rich motif GXXGXXA. Within this consensus, they have redidues corresponding to N_(21) and L_(22) of AtBAN which may determine substrate specificities. Besides, they also have a perfect leucine zipper like AtBAN.
They share very similar secondary structures.αhelix is the most abundant proportion (39.7742.60% by frequency), followed by random coil (30.04-32.84%), supplemented with a little extended strand andβ-tum. They have 2 bigαhelices at the middle of protein and 1 bigαhelix near-C-terminus. The also share very similar tertiary structure, resembling Dihydroflavonol reductase from Vtiis.Vinifera in general, and partilly similar to NADPH-Dependent carbonyl reductase from Sporobolomyces.Salmonicolor at local regions.
Clues from gene structure, protein structure and sequence identities all suggest that BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 are orthologous genes of AtBAN.
3) B. rapa and B. oeracea are donors of genetic materials for B. napus
BnBAN-1 and BoBAN-1 share 97.5% and 99.1% of identities on whole genomic sequence and ORF scales respectively, and 96.5% and 99.5% between BnBAN-2 and BoBAN-2. These homologies are much higher than that between intra-species paralogs BnBAN-1/BnBAN—3 and BnBAN-2. On phylogenetic trees of both nucleotide and amino acid sequences, BnBAN-1 groups with BoBAN-1 first, and BnBAN-2 groups with BoBAN-2 first Furhermore, clues from intron similarities and featured mutation sites on both nucleotide and amino acid sequences, all point to the corresponding relationships of BnBAN-1 to BoBAN-1 and BnBAN-2 to BoBAN-2, suggesting that B. rapa and B. oeracea have provided the genetic material for B. napus. This research provided straight and concrete evidence for revealing me evolutionary relationships among B. napus, B. rapa and B. oeracea, based on a profile of comparative cloning of full-length functional gene family.
4) Number of members of BnBAN gene family, BrBAN gene family and BoBAN gene family
The genomic DNA of line 5B was subjected to EcoRI, EcoRV, SphI, SacI and XbaI digestion respectively, and Southern blot result indicated that there may be at least 4 members of BnBAN family in B. napus. Because we haven't accomplished the cloning of the whole BAN gene sets from these 3 species, and Southern hybridization of the 2 prental species is still lack, so further exhaustive cloning is neccessary to identify whether the BAN locus has been triplicated in basic "diplioid" Brassica species as compared with A. thaliana.
甘蓝型油菜黄籽性状形成的分子机理目前还不清楚，但可以借鉴同属于十字花科的拟南芥透明种皮(TRANSPARENT TESTA，TT)性状研究的成果。花青素苷和缩合单宁的合成共用的是一种底物，而由BANYULS(BAN)基因编码的花青素还原酶(Anthocyanidin reductase，ANR)正是在类黄酮物质合成途径中决定这种底物是否转向缩合单宁合成的关键酶。BAN将花青素还原为2，3-顺式-黄酮-3-醇的功能，随后2，3-顺式-黄酮-3-醇聚合成为缩合单宁。研究甘蓝型油菜及其2个亲本物种的BAN基因有助于进一步研究甘蓝型油菜黄籽性状形成的分子机制，为通过遗传工程创造稳定的甘蓝型黄籽油菜新材料提供基础。
本研究克隆了甘蓝型油菜BAN基因家族(BnBAN)的3个成员、白菜型油菜BAN基因家族(BrBAN)的2个成员和甘蓝BAN基因家族的2个成员(BoBAN)的cDNA序列及其基因组全长序列，并进行了系统分析，主要结果如下：
1)率先克隆了甘蓝型油菜及其2个亲本物种的BAN基因家族
本研究利用RACE技术，率先获得了甘蓝型油菜及其亲本物种白菜型油菜和甘蓝的BAN基因家族部分成员全长eDNA和对应的基因组序列。BnBAN-1、BnBAN-2和BnBAN-3分别为1571 bp、1612 bp和1574 bp，推导的mRNA分别为1178 bp、1124 bp和1180 bp(不计poly(A)尾巴，下同)；BrBAN-1和BrBAN-2分别为1607 bp和1623 bp，推导的mRNA分别为1212 bp和1174 bp；BoBAN-1和BoBAN-2分别为1543 bp和1642 bp，推导的mRNA分别为1172 bp和1150 bp。这是在芸薹属植物中首次克隆全长BAN基因，为深入研究芸薹属植物BAN基因的功能、进化、调控模式奠定了基础。
2)芸薹属BAN基因具有BAN的典型特征
芸薹属7个BAN基因都有5个内含子和6个外显子，符合GT……AG的内含子边界序列特征，并且和AtBAN的位置相符。在BnBAN-1、BnBAN-2、BnBAN-3、BrBAN-1、BrBAN-2、BoBAN-1和BoBAN-2 mRNA的41-1069、50-1066、41-1078、74-1111、46-1062、47-1063、41-1069 bp区段有分别为1029、1017、1038、1038、1017、1017、1017 bp的开放阅读框(ORF，包括终止密码子)，5’UTR分别为40、49、40、73、45、46、40 bp，3’UTR分别为109、58、102、101、112、87、103 bp。分别在它们polyA加尾位点上游的22-42、23-43、22-42、20-39、20-42、15-34、22-45 bp有可能的非典型polyA加尾信号TATAAA和AAATAAT。
BnBAN-1、BnBAN-2、BnBAN-3、BrBAN-1、BrBAN-2、BoBAN-1和BoBAN-2分别编码由342、338、345、345、338、342、338个氨基酸组成的多肽链，理论分子量(Mw)分别为37.850、37.680、38.277、38.266、37.614、37.927和37.554，等电点(pI)分别为6.38、4.93、6.38、6.06、5.00、5.81和4.87。它们都编码酸性蛋白，氨基酸组成中丝氨酸的含量均为最高。BnBAN-1、BnBAN-3和BrBAN-1有24个潜在的磷酸化位点，BnBAN-2、BrBAN-2和BoBAN-2都有18个，BoBAN-1有23个。预测它们均没有信号肽和跨膜域，可能象AtBAN一样定位于细胞质中。
芸薹属3个物种的7个BAN蛋白与AtBAN和苜蓿BAN一样，也具有典型的罗斯曼折叠，表现在都具有一个保守的富含谷氨酸的序列GXXGXXA，在该序列中它们均有同已报道的拟南芥中决定BAN底物特异性的氨基酸N_(21)和L_(22)对应的残基，同时在这7个蛋白均存在一个与AtBAN高度相似的亮氨酸拉链。它们的二级结构非常相似，以α螺旋所占比例最高，占氨基酸总数的39.77～42.60％，随机卷曲占30.04～32.84％，有少量的延伸链和β转角。它们蛋白的中部都有2个大的α螺旋，C末端也有1个。它们的三级结构也很相似，与葡萄黄烷酮醇还原酶的晶体整体相似，与赭色掷孢酵母的NADPH依赖型羟基还原酶部分相似。
从基因结构、蛋白结构、序列同源性三大方面分析，均显示芸薹属3个物种的7个BAN基因是AtBAN的垂直同源基因，具有BAN的典型特征。
3)甘蓝、白菜型油菜为甘蓝型油菜提供了遗传物质
BnBAN-1和BoBAN-1之间基因组序列和编码区序列的一致性分别高达97.5％和99.1％。BnBAN-2和BoBAN-2之间基因组序列和编码区序列的一致性分别高达96.5％和99.5％。这些种间基因的一致性高于甘蓝型油菜种内成员之间的一致性。无论核酸水平还是氨基酸水平的系统发生学聚类结果，内含子的相似度，基因序列和氨基酸序列上的特异位点均显示，BnBAN-1和BoBAN-1紧密地聚在一起，BnBAN-2和BoBAN-2紧密地聚在一起。可以确定BnBAN-1和BnBAN-2来自于甘蓝(BoBAN-1和BoBAN-2)。甘蓝和白菜型油菜的确为甘蓝型油菜提供了遗传物质。本研究从编码关键酶的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。
4)甘蓝型油菜、白菜型油菜和甘蓝BAN基因家族的成员数
采用甘蓝型油菜典型黑籽系5B的基因组DNA为材料，分别用EcoRI、EcoRV、SphI、SacI、XbaI这5种限制性内切酶进行酶切和Southern blot杂交，结果显示BnBAN家族可能存在4个以上成员。由于这3个物种BAN基因家族全部成员克隆工作尚未完成，且没有对白菜型油菜和甘蓝的BAN基因进行Southern杂交，因此有待于进一步的进行竭尽式的克隆，才能回答芸薹属二倍体基因种相对于拟南芥在BAN功能位点究竟是否符合典型的“三倍化”模型。
Oilseed rape (Brassica napus) is one of the most important oil crops. Its yellow-seeded type shows many quality advantages, and yellow seed trait is a hotspot of rapeseed breeding goal worldwide. Both parental speices, B. oeracea and B. rapa, have stable-phenotyped natural yellow seed genotypes, but B. napus has not. Yellow seed trait introgression through 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 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 molecular mechanism of yellow seed trait formation is not clear still, but achievements on the TRANSPARENT TESTA (TT) trait in Arabidopsis thaliana can be referred The anthocyanins and the condensed tannin use common substrate. The BANYULS (BAN) gene coding anthocyanidin reductases (ANR) determines whether the substrate enters the condensed tannin synthesis pathway. BAN reducecs the anthocyanidin to 2,3-cis-flavan-3-ols which are then polymerized later to form condensed tannin. Study on BAN genes from B. napus and its parental species will help to reveal the molecular mechanism of yellow seed trait formation and lay the base for transgenetic creation of stably inherited yellow seed stocks.
In this research, the full-length cDNAs and corresponding genomic sequences of 3 members of B. napus BAN gene family (BnBAN), 2 members of B. rapa BAN gene family (BrBAN) and 2 members of B. oeracea BAN gene family (BoBAN) were isolated and systemically analysized. The main results are as follows:
1) Cloning of BAN genes from B. napus and its parental species
Using rapid amplification of cDNA ends (RACE) technology, full-length cDNAs and corresponding genomic sequences of BAN genes from B. napus and its parental species were isolated BnBAN-1, BnBAN-2 and BnBAN-3 are 1571,1612 and 1574 bp, with the corresponding mRNAs of 1178,1124 and 1180 bp (not including poly A tail), respectively. BrBAN-1 and BrBAN-2 are 1607 and 1623 bp, with the corresponding mRNAs of 1212 and 1174 bp, respectively. BoBAN-1 and BoBAN-2 are 1543 and 1642 bp, with the corresponding mRNAs of 1172 and 1150 bp, respectively. This is first report of full-length BAN gene cloning from Brassica species, and will lay the base for study of function, evolution, regulatory mode of BAN genes of Brassica.
2) BnBAN,BrBAN and BoBAN conform to typical features of BAN
They all have 5 introns with the same positions as those of AtBAN. All the introns conform to canonical intron splicing boundary fearture sequence "GT...AG". At 41-1069, 50-1066, 41-1078, 74-1111,46-1062,47-1063 and 41-1069 bp of BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 mRNAs have an open reading frame (ORF) of 1029,1017,1038,1038,1017,1017 and 1017bp (including stop codon). Their 5' UTRs are 40,49,40,73,45,46 and 40 bp, and 109,58,102,101,112,87 and 103 bp for 3' UTRs, respectively. Their possible non-canonical poly A tailing signals TATAAA and AAATAAT are located 22-42,2343,22-42,20-39,20-42,15-34 and 22-45 bp respectively upstream the poly A tailing sites.
The deduced BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 proteins are 342,338,345,345,338,342 and 338 aa in length, with Mw of 37.850,37.680,38.277,38.266,37.614,37.927 and 37.554 kDa, and pI of 6.38,4.93,6.38,6.06,5.00,5.81 and 4.87, respectively. They all are typical acidic proteins. Serine is the richest one in their amino acid compositions. BnBAN-1, BnBAN-3 and BrBAN-1 have 24 potential phosphorylation sites; BnBAN-2, BrBAN-2 and BoBAN-2 have 18; and BoBAN-1 has 23. They were predicted with no signal peptide and transmembrane domain, with the higheset possibility to be located in cytoplasm.
Like AtBAN and MtBAN, the 7 Brassica BAN proteins have classical Rossmann fold characterized by a conserved glycine-rich motif GXXGXXA. Within this consensus, they have redidues corresponding to N_(21) and L_(22) of AtBAN which may determine substrate specificities. Besides, they also have a perfect leucine zipper like AtBAN.
They share very similar secondary structures.αhelix is the most abundant proportion (39.7742.60% by frequency), followed by random coil (30.04-32.84%), supplemented with a little extended strand andβ-tum. They have 2 bigαhelices at the middle of protein and 1 bigαhelix near-C-terminus. The also share very similar tertiary structure, resembling Dihydroflavonol reductase from Vtiis.Vinifera in general, and partilly similar to NADPH-Dependent carbonyl reductase from Sporobolomyces.Salmonicolor at local regions.
Clues from gene structure, protein structure and sequence identities all suggest that BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 are orthologous genes of AtBAN.
3) B. rapa and B. oeracea are donors of genetic materials for B. napus
BnBAN-1 and BoBAN-1 share 97.5% and 99.1% of identities on whole genomic sequence and ORF scales respectively, and 96.5% and 99.5% between BnBAN-2 and BoBAN-2. These homologies are much higher than that between intra-species paralogs BnBAN-1/BnBAN—3 and BnBAN-2. On phylogenetic trees of both nucleotide and amino acid sequences, BnBAN-1 groups with BoBAN-1 first, and BnBAN-2 groups with BoBAN-2 first Furhermore, clues from intron similarities and featured mutation sites on both nucleotide and amino acid sequences, all point to the corresponding relationships of BnBAN-1 to BoBAN-1 and BnBAN-2 to BoBAN-2, suggesting that B. rapa and B. oeracea have provided the genetic material for B. napus. This research provided straight and concrete evidence for revealing me evolutionary relationships among B. napus, B. rapa and B. oeracea, based on a profile of comparative cloning of full-length functional gene family.
4) Number of members of BnBAN gene family, BrBAN gene family and BoBAN gene family
The genomic DNA of line 5B was subjected to EcoRI, EcoRV, SphI, SacI and XbaI digestion respectively, and Southern blot result indicated that there may be at least 4 members of BnBAN family in B. napus. Because we haven't accomplished the cloning of the whole BAN gene sets from these 3 species, and Southern hybridization of the 2 prental species is still lack, so further exhaustive cloning is neccessary to identify whether the BAN locus has been triplicated in basic "diplioid" Brassica species as compared with A. thaliana.
引文
安彩泰，马静芳．油菜生物化学．甘肃民族出版社，1993，pp 214-217．
陈宝元，孟金陵．甘蓝型油菜(Brassica napus L.)种皮发育的初步观察[J]．华中农学院学报，1984，2：5-8．
李殿荣，田建华．甘蓝型油菜显性黄籽基因的发现和黄籽种的选育．中国作物学会油料专业委员会第四次全国代表大会学术论文摘要．2000，30-31．
李加纳，张学昆，谌利，等．不同遗传背景的甘蓝型黄籽油菜粒色遗传初步研究．中国油料作物学报，1998，20(4)：16-19．
李运涛，李加纳，柴友荣，等．Cloning and Sequence Analysis of a DFR Gene from Brassica campestris L. var. oleifera DC．分子植物育种，2005，3(4)：485-492．
粱艳丽，梁颖，李加纳，等．甘蓝型黄、黑籽油菜种皮特性比较研究．中国油料作物学报，2002，24(4)：14-17．
刘后利，韩继祥．中国黄籽泊菜种质资源的现状及其研究和利用前景．刘后利科学论文集．北京：北京农业大学出版社，1994．
刘后利．甘蓝型黄籽油菜的发现及其遗传行为的初步研究．遗传学报，1979，6(1)：54．
刘后利．甘蓝型黄籽油菜的发现遗传行为的初步研究．遗传学报，1979，6(1)：54．
刘后利．甘蓝型黄籽油菜的遗传研究．作物学报，1992，18(4)：241-249．
刘后利．油菜遗传育种学．北京：中国农业大学出版社，2000，104-108，220-225．
刘英，王超．简述甘蓝类植物的起源与分类．北方园艺，2006，4：57．
刘玉梅．方智远，孙培田．我国甘蓝遗传育种的研究进展和预测．中国蔬菜，1997，(6)：1-3．
王汉中，刘后利．油菜皮壳中多酚氧化酶活性的组织化学检定及其与粒色的关系．中国油料，1991，(1)：30-33．
吴江生，石淑稳．甘蓝型黄籽突变体的遗传研究．中国油料作物学报，1998，20(3)：5-9．
叶小利，李家纳，唐章林，等．甘蓝型油菜种皮色泽及相关性状的研究．作物学报，2001，27(5)：550-556．
赵志伟，曾凡亚，赵云，等．甘蓝型油菜BAN同源基因片段克隆与序列分析．中国油料作物学报，2001，23(4)：7-10．
Abraham V, Bhatia C R. Development of strains with yellow-seed coat in Indian mustard(Brassica juncea Czern & Coss). Plant Breed, 1986, 97: 86-88.
Bendtsen J D, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol, 2004, 340: 783-795.
Bogs J, Downey M, Harvey J S, et al. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol, 2005, 139: 652-663.
Caboche M, Lepiniec L. The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat. Plant Cell, 2002, 14: 2463-2479.
Chapple C C S, Shirley B W, Zook M, et al. Secondary metabolism in Arabidopsis, in ARABIDOPSIS, Cold Spring Harbor Press (C. Somerville and E. Meyerowitz, eds.), 1994, pp 989-1030.
Chen A H, Chai Y R, Li J N, et al. Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus). J Biochem Mol Biol, 2007, 40(2): 247-260.
Debeaujon I, Karen M L, Maarten K. Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis. Plant Physiol, 2000, 122: 403-413.
Debeaujon I, Nesi N, Perez P, et al. Proanthoeyanidin-accumulating cells in Arabidopsis testa: Regulation of differentiation and role in seed development. Plant Cell, 2003, 15(11): 2514-2531.
Debeaujon I, Peeters A J, Léon-Kloosterziel K M, et al. The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell, 2001, 13: 853-871.
Devic M, Guilleminot J, Debeaujon I, et al. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Plant J, 1999, 19(4): 387-398.
Feinbaum R L, Ausubel F M. Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol Cell Biol, 1998, 8: 1985-1992.
Focks N, Sagasser M, Weisshaar B, et al. Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana. Planta, 1995, 208: 352-357.
Geourjon C, Deléage G SOPMA: significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci, 1995, 11: 681-684.
Getinet A, Rakow G. Repression of seed coat pigmentation in Ethiopian mustard. Can J Plant Sci, 1997, 77: 501-505.
Hofmann K, Stoffel W. TMbase-A database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler, 1993, 374: 166.
Johnston J S, Pepper A E, Hall A E, et al. Evolution of genome size in Brassicaceae. Ann Bot, 2005, 95: 229-235.
Koornneef M, Joma M L, van der Swan D L C, et al. The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana Heynh. Theor Appl Genet, 1982, 61: 385-395.
Lagercrantz U. Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements. Genetics, 1998, 150: 1217-1228
Leon-Kloosterziel K M, Keijzer C J, Koornneef M. A seed shape mutant of Arabidopsis that is affected in integument development. Plant Cell, 1994, (6): 3895-392.
Marchler-Bauer A, Bryant S H. CD-Search: protein domain annotations on the fly. Nucleic Acids Res, 2004, 32: W327-W331.
McCugan J M. Seeds and seeding of the genus Brassica. Canade J Res, 1948, 26:
Mitaku S, Hirokawa T, Tsuji T. Amphiphilicity index of polar amino acids as an aid in the characterization of amino acid preference at membrane-water interfaces. Bioinformatics, 2002, 18: 608-616.
Mohammad A, Sikka S M, Aziz M A. Inheritance of seed colour in some oleiferous Brassicae. Indian J Genet Breed, 1942, 2: 112-127.
Olsson G. Species crosses within the genus Brassica. Artificial Brassica napus L. Hereditas, 1960, (46): 351-396.
Pelletier M K, Shidy B W. Analysis of Flavanone 3'-Hydroxylase in Aradidopsis thaliana seedling. Plant Physiol, 1996, 111: 339-345.
Punyasiri P A N, Abeysinghe I S B, Kumar V, et al. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways [J]. Arch Biochem Biophy, 2004, 431: 22-30.
Schmidt R, Acarkan A, Boivin K. Comparative structural genomics in the Brassicaceae family. Plant Physiol Biochem, 2001, 39: 253-262.
Schoenbohm C, Martens S, Eder C, et al. Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450 enzyme. Biol Chem, 2000, 381: 749-753.
Shirley B W, Hanley S, Goodman H M. Effects of ionizing radiation on a plant genome: Analysis of two Arabidopsis transparent testa mutations. Plant Cell, 1992, 4: 333-347
Shirley B W, Kubasek, W L, Storz G, et al. Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. Plant J, 1995, 8: 659-671.
Shirzadegan M, Robbelen G. Influence of seed color and hull proportion on quality properties of seed in Brassica napus L. Fette Seifen Anstrichmittel, 1985: 235-237.
Tanaka Y, Tano S, Chantes T, et al. A new Arabidopsis mutant induced by ion beams affects flavonoid synthesis with spotted pigmentation in testa. Genes Genet Syst, 1997, 72, 141: 148
Tang Z L, Li J N. Genetic variation of yellow-seeded rapeseed lines (B. napus L.) from different genetic sources. Plant Breed, 1996, 116(5): 471-474.
Tusnády G E, Simon I. The HMMTOP transmembrane topology prediction server. Bioinformatics, 2001, 17: 849-850.
UN Genome analysis in Brassica with special reference to the experimental formation of B. napus and the peculiar mode of fertilization. Japn J Bot 1935, 7: 389-452.
Wei Y L, Li J N, Lu J, et al. Molecular cloning of Brassica napus TRANSPARENT TESTA 2 gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis. Mol Biol Rep, 2007, 34(2): 105-120.
Wisman E, Hartmann U, Sagasser M, et al. Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc Natl Acad Sci USA, 1998, 95: 12432-12437.
Xie DY, Sharma S B, Paiva N L, et al. Role of anthoeyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science, 2003, 299: 396-399.
Xu B B, Li J N, Zhang X K, et al. Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus. J Plant Physiol, 2007, 164(3): 350-363
陈宝元，孟金陵．甘蓝型油菜(Brassica napus L.)种皮发育的初步观察[J]．华中农学院学报，1984，2：5-8．
李殿荣，田建华．甘蓝型油菜显性黄籽基因的发现和黄籽种的选育．中国作物学会油料专业委员会第四次全国代表大会学术论文摘要．2000，30-31．
李加纳，张学昆，谌利，等．不同遗传背景的甘蓝型黄籽油菜粒色遗传初步研究．中国油料作物学报，1998，20(4)：16-19．
李运涛，李加纳，柴友荣，等．Cloning and Sequence Analysis of a DFR Gene from Brassica campestris L. var. oleifera DC．分子植物育种，2005，3(4)：485-492．
粱艳丽，梁颖，李加纳，等．甘蓝型黄、黑籽油菜种皮特性比较研究．中国油料作物学报，2002，24(4)：14-17．
刘后利，韩继祥．中国黄籽泊菜种质资源的现状及其研究和利用前景．刘后利科学论文集．北京：北京农业大学出版社，1994．
刘后利．甘蓝型黄籽油菜的发现及其遗传行为的初步研究．遗传学报，1979，6(1)：54．
刘后利．甘蓝型黄籽油菜的发现遗传行为的初步研究．遗传学报，1979，6(1)：54．
刘后利．甘蓝型黄籽油菜的遗传研究．作物学报，1992，18(4)：241-249．
刘后利．油菜遗传育种学．北京：中国农业大学出版社，2000，104-108，220-225．
刘英，王超．简述甘蓝类植物的起源与分类．北方园艺，2006，4：57．
刘玉梅．方智远，孙培田．我国甘蓝遗传育种的研究进展和预测．中国蔬菜，1997，(6)：1-3．
王汉中，刘后利．油菜皮壳中多酚氧化酶活性的组织化学检定及其与粒色的关系．中国油料，1991，(1)：30-33．
吴江生，石淑稳．甘蓝型黄籽突变体的遗传研究．中国油料作物学报，1998，20(3)：5-9．
叶小利，李家纳，唐章林，等．甘蓝型油菜种皮色泽及相关性状的研究．作物学报，2001，27(5)：550-556．
赵志伟，曾凡亚，赵云，等．甘蓝型油菜BAN同源基因片段克隆与序列分析．中国油料作物学报，2001，23(4)：7-10．
Abraham V, Bhatia C R. Development of strains with yellow-seed coat in Indian mustard(Brassica juncea Czern & Coss). Plant Breed, 1986, 97: 86-88.
Bendtsen J D, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol, 2004, 340: 783-795.
Bogs J, Downey M, Harvey J S, et al. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol, 2005, 139: 652-663.
Caboche M, Lepiniec L. The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat. Plant Cell, 2002, 14: 2463-2479.
Chapple C C S, Shirley B W, Zook M, et al. Secondary metabolism in Arabidopsis, in ARABIDOPSIS, Cold Spring Harbor Press (C. Somerville and E. Meyerowitz, eds.), 1994, pp 989-1030.
Chen A H, Chai Y R, Li J N, et al. Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus). J Biochem Mol Biol, 2007, 40(2): 247-260.
Debeaujon I, Karen M L, Maarten K. Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis. Plant Physiol, 2000, 122: 403-413.
Debeaujon I, Nesi N, Perez P, et al. Proanthoeyanidin-accumulating cells in Arabidopsis testa: Regulation of differentiation and role in seed development. Plant Cell, 2003, 15(11): 2514-2531.
Debeaujon I, Peeters A J, Léon-Kloosterziel K M, et al. The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell, 2001, 13: 853-871.
Devic M, Guilleminot J, Debeaujon I, et al. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Plant J, 1999, 19(4): 387-398.
Feinbaum R L, Ausubel F M. Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol Cell Biol, 1998, 8: 1985-1992.
Focks N, Sagasser M, Weisshaar B, et al. Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana. Planta, 1995, 208: 352-357.
Geourjon C, Deléage G SOPMA: significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci, 1995, 11: 681-684.
Getinet A, Rakow G. Repression of seed coat pigmentation in Ethiopian mustard. Can J Plant Sci, 1997, 77: 501-505.
Hofmann K, Stoffel W. TMbase-A database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler, 1993, 374: 166.
Johnston J S, Pepper A E, Hall A E, et al. Evolution of genome size in Brassicaceae. Ann Bot, 2005, 95: 229-235.
Koornneef M, Joma M L, van der Swan D L C, et al. The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana Heynh. Theor Appl Genet, 1982, 61: 385-395.
Lagercrantz U. Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements. Genetics, 1998, 150: 1217-1228
Leon-Kloosterziel K M, Keijzer C J, Koornneef M. A seed shape mutant of Arabidopsis that is affected in integument development. Plant Cell, 1994, (6): 3895-392.
Marchler-Bauer A, Bryant S H. CD-Search: protein domain annotations on the fly. Nucleic Acids Res, 2004, 32: W327-W331.
McCugan J M. Seeds and seeding of the genus Brassica. Canade J Res, 1948, 26:
Mitaku S, Hirokawa T, Tsuji T. Amphiphilicity index of polar amino acids as an aid in the characterization of amino acid preference at membrane-water interfaces. Bioinformatics, 2002, 18: 608-616.
Mohammad A, Sikka S M, Aziz M A. Inheritance of seed colour in some oleiferous Brassicae. Indian J Genet Breed, 1942, 2: 112-127.
Olsson G. Species crosses within the genus Brassica. Artificial Brassica napus L. Hereditas, 1960, (46): 351-396.
Pelletier M K, Shidy B W. Analysis of Flavanone 3'-Hydroxylase in Aradidopsis thaliana seedling. Plant Physiol, 1996, 111: 339-345.
Punyasiri P A N, Abeysinghe I S B, Kumar V, et al. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways [J]. Arch Biochem Biophy, 2004, 431: 22-30.
Schmidt R, Acarkan A, Boivin K. Comparative structural genomics in the Brassicaceae family. Plant Physiol Biochem, 2001, 39: 253-262.
Schoenbohm C, Martens S, Eder C, et al. Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450 enzyme. Biol Chem, 2000, 381: 749-753.
Shirley B W, Hanley S, Goodman H M. Effects of ionizing radiation on a plant genome: Analysis of two Arabidopsis transparent testa mutations. Plant Cell, 1992, 4: 333-347
Shirley B W, Kubasek, W L, Storz G, et al. Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. Plant J, 1995, 8: 659-671.
Shirzadegan M, Robbelen G. Influence of seed color and hull proportion on quality properties of seed in Brassica napus L. Fette Seifen Anstrichmittel, 1985: 235-237.
Tanaka Y, Tano S, Chantes T, et al. A new Arabidopsis mutant induced by ion beams affects flavonoid synthesis with spotted pigmentation in testa. Genes Genet Syst, 1997, 72, 141: 148
Tang Z L, Li J N. Genetic variation of yellow-seeded rapeseed lines (B. napus L.) from different genetic sources. Plant Breed, 1996, 116(5): 471-474.
Tusnády G E, Simon I. The HMMTOP transmembrane topology prediction server. Bioinformatics, 2001, 17: 849-850.
UN Genome analysis in Brassica with special reference to the experimental formation of B. napus and the peculiar mode of fertilization. Japn J Bot 1935, 7: 389-452.
Wei Y L, Li J N, Lu J, et al. Molecular cloning of Brassica napus TRANSPARENT TESTA 2 gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis. Mol Biol Rep, 2007, 34(2): 105-120.
Wisman E, Hartmann U, Sagasser M, et al. Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc Natl Acad Sci USA, 1998, 95: 12432-12437.
Xie DY, Sharma S B, Paiva N L, et al. Role of anthoeyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science, 2003, 299: 396-399.
Xu B B, Li J N, Zhang X K, et al. Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus. J Plant Physiol, 2007, 164(3): 350-363