甘蓝型油菜（Brassica napus L.）TT8基因家族的克隆及反义转化
摘要
甘蓝型油菜(Brassica napus L．)是世界四大油料作物之一，也是我国重最要的油料作物之一。黄籽油菜近年颇受重视，与黑籽油菜相比，具有种皮色素少、种皮薄、纤维素含量低以及蛋白质和含油量高的优点，培育遗传稳定、农艺性状优良的黄籽甘蓝型油菜已成为油菜育种的一个重要目标之一。甘蓝型油菜黄籽性状形成的分子机理目前还不清楚，但可以借鉴拟南芥透明种皮(Transparent testa，TT)性状研究成果，开展甘蓝型油菜种皮色泽分子机制的研究。目前在拟南芥中已鉴定出21个TT基因位点和一个BAN位点，并且许多TT基因在拟南芥中已经被克隆。
拟南芥TT8(AtTT8)是类黄酮合成途径中的一个调控因子，它属于碱性螺旋-环-螺旋(basic-Helix-loop-helix domain，bHLH)转录因子，此类转录因子在生物的发育过程中具有重要作用，拟南芥、玉米、矮牵牛、金鱼草等植物中该基因的突变导致种子颜色或花色改变。拟南芥中，TT8、TTG1、TT2三个蛋白都调控BAN基因和DFR基因的转录。研究油菜TT8基因有助于进一步了解甘蓝型油菜黄籽性状形成的分子机制，可以为通过基因工程创造稳定的黄籽甘蓝型油菜新材料提供基础。
本研究克隆了甘蓝型油菜TT8基因家族的3个成员的全长cDNA和1个成员的基因组序列，并对其序列及编码蛋白进行了系统的分析；通过Sourthern杂交鉴定了TT8基因家族在甘蓝型油菜中的成员数；构建了甘蓝型油菜TT8基因家族反义植物表达载体，并转化黑籽甘蓝型油菜得到了转基因植株。主要结果如下：
1、甘蓝型油菜BnTT8基因家族3个成员的克隆和分析
根据AtTT8保守区设计引物，以甘蓝型油菜黑籽系5B生殖器官总RNA为材料，利用RACE(rapid amplification of cDNA ends)技术，克隆了BnTT8基因家族的3个成员的全长cDNA序列，分别命名为BnTT8-1、BnTT8-2、BnTT8-3，并克隆了BnTT8-1的基因组序列。
BnTT8-1基因组序列长2949bp，含6个内含子，mRNA(不包括polyA，下同)为1771bp，开放阅读框(ORF，包括终止密码子，下同)为1566bp，5’UTR为147bp，3’UTR为58bp。BnTT8-2mRNA为1783bp，ORF为1566bp，5’UTR为137bp，3’UTR为80bp。BnTT8-3 mRNA为1676bp，的ORF为1308bp，5’UTR为134bp，3’UTR为231bp。在BnTT8-1、BnTT8-2和BnTT8-3在3’UTR均存在典型的AATAAA加尾信号，在5’UTR区都有富含AG重复的微卫星序列。核苷酸组成在不同区段变化较大，非编码区G+C含量明显低于编码区。BnTT8-1的内含子的总长度比拟南芥大大缩短。
推导的BnTT8-1蛋白为521个氨基酸，Mw=59.669kDa，pI=5.61；BnTT8-2为1个521个氨基酸，Mw=59.955kDa，pI=5.65；BnTT8-3为435个氨基酸，Mw=50.071kDa，pI=5.47。3个蛋白中均以谷氨酸的含量为最高，且酸性氨基酸数目多于碱性的氨基酸。BnTT8-3 mRNA对应于其它TT8基因的终止密码子发生突变，且与其它TT8 mRNA相比在该位置上游发生了一段107bp的序列缺失，导致编码的蛋白比其它TT8蛋白在C-末端缺少86个氨基酸。
BnTT8-1、BnTT8-2和BnTT8-3之间在氨基酸水平上有很高的同源性，BnTT8-1和BnTT8-2的一致性和相似性分别为96.7％和98.1％，BnTT8-1和BnTT8-3的一致性和相似性分别为97％和98.6％，BnTT8-2和BnTT8-3的一致性和相似性分别为98.4％和99.3％。BnTT8基因的核酸及蛋白序列与已知植物的TT8基因和bHLH基因的核酸及蛋白序列有高度的同源性，尤与AtTT8最相似。BnTT8-1与AtTT8的相似性为83.4％，一致性为76.5％；BnTT8-2与AtTT8的相似性为83.9％，一致性为76.9％；BnTT8-3与AtTT8的相似性为83.9％，一致性为77.5％。BnTT8-1、BnTT8-2和BnTT8-3蛋白都在L_(366)到H_(417)区域分别对应存在HLH保守性结构域，在该结构域及保守性氨基酸位置，它们与AtTT8高度相似。它们分别有33个、33个和32个潜在的磷酸化位点，说明磷酸化作用可能与它们的活性有关。它们的二级结构主要由a螺旋和随机卷曲组成，延伸链和β转角也占一定比例。
BnTT8家族3个成员可能和AtTT8一样，调控类黄酮途径的结构基因表达。由于BnTT8-3蛋白的C-末端缺失了86个氨基酸，所以该成员蛋白是否具有活性，活性强弱如何，值得进一步研究。
2、甘蓝型油菜BnTT8基因家族的成员数
Southern blot杂交结果中，每种限制酶均获得3条或3条以下的杂交条带。而本研究采用竭尽式RACE克隆方式，也只获得了3个成员的全长cDNA。因此推测，甘蓝型油菜中BnTT8基因家族可能只存在3个成员。
3、BnTT8反义表达载体的构建
将BnTT8基因家族共保守的817bp的片段反接到中间表达载体pCambia2301G中，替换其上的GUS基因，形成CaMV 35S启动子驱动、反义共抑制所有BnTT8成员的植物表达载体，命名为pFBnTT8A。
4、甘蓝型油菜TT8反义转化植株的获得
采用农杆菌介导法转化甘蓝型油菜“湘油15”，获得了24株抗卡那霉素的再生苗，通过检测GUS基因在抗性植株中的稳定表达情况，发现其中13株为稳定表达的转基因植株，转化频率达到2.23％。
Oilseed rape (Brassica napus L.) is one of the four major oil crops in the world and is one of the most important oil plants in China too. In recent year the yellow-seeded B. napus has attracted researchers' interest in that it has many good properties compared to black seeds, such as thinner seed coat, fewer seed coat pigment, lower meal fiber content, higher seed oil content and meal protein content, etc. It is one of the most important objectives to breed yellow-seeded rapeseed cultivars with stably inherited yellow seed trait and good agronomic traits. The molecular mechanism of the yellow seed trait is still unclear in B. napus. However, the achievements on the Transparent Testa (TT) trait in Arabidopsis thaliana are available for an insight into the formation of the yellow seed color in B. napus. Up to now, 21 TT loci and a BAN locus have been identified, and many of the TT genes have been cloned in A thaliana.
The A. thaliana TT8 (AtTT8) gene encodes a regulative factor in the flavanone synthesis way. The AtTT8 belongs to the basic helix-loop-helix domain (bHLH) transcription factors which play an important role in the biologic development. In A thaliana, Zea mays, Petunia hybrida, Antirrhinum majus and so on, the TT8 (or analogue) gene mutation alters seed color or flower color. In A. thaliana, the TT8, TTG1 and TT2 proteins all regulate the BAN and DFR genes' transcription. Study on B. napus TT8 gene is helpful to understand the molecular mechanism of the yellow seed color formation and may lay a foundation for creating stable yellow-seeded lines by genetic engineering.
In this research, full-length cDNAs of 3 members of B. napus TT8 gene family (BnTT8) were isolated, together with genomic sequence of one member. The nucleotide sequences and the corresponding polypeptides of the BnTT8 gene family were systematically analyzed. The mumber of the BnTT8 gene family members was identified through Sourthern-blot. A BnTT8 gene family antisense plant expression vector was constructed and some regenerated plantlets were obtained from black-seeded B. napus explant cultivar transformed with this vector.
1. Cloning and analysis of the 3 members of BnTT8 gene family in B. napus
Based on the conserved regions of the A. thaliana TT8 gene, the PCR pimers were designed. 3 full-length of cDNA sequences of the B. napus TT8 gene famliy were cloned using total RNA from reproductive organs of black-seeded B.napus line 5B by RACE (rapid amplification of cDNA ends) technique. They were named as BnTT8-1, BnTT8-2 and BnTT8-3, respectively. Furthermore, the genomic sequence of BnTT8-1 was also cloned.
BnTT8-1 has a genomic sequence of 2 949bp with 6 introns. Its mRNA (excluding the polyA tail) is 1 771 bp, with an open reading frame (ORF, including the stop codon) of 1 566bp. Its 5' UTR and 3' UTR are 147bp and 58bp respectively. BnTT8-2 mRNA is 1 783bp, with an ORF of 1 566bp, a 5' UTR of 137bp, and a 3' UTR of 80bp. BnTT8-3 mRNA is 1 676bp, with an ORF of 1 308bp, a 5' UTR of 137bp and a 3' UTR of 231bp. There is a typical polyadenylation signal AATTAAA in 3' UTR of them, and there are microsattilites rich in AG-repeats in their 5' UTRs. The nucleotide composition is varied in different regions of them. The G+C content of the non-coding region is obviously lower than that of the coding region. In BnTT8-1, the total intron length is much shorter than A. thaliana.
The deduced BnTT8-l protien is 521 aa, Mw=59.669 kDa, pI=5.61; BnTT8-2 is also 521 aa, Mw=59.955 kDa, pI=5.65; and BnTT8-3 is 435 aa, Mw=50.071 kDa, pI=5.47. Glutamic acid is the most abundant residue in them, and acidic residues are more than basic ones. BnTT8-3 mRNA shows mutation at position corresponding stop codon of typical TT8 genes, and a 107-bp deletion exists just upstream this site, causing the loss a 86-aa C-terminal region in its deduced protein.
BnTT8-1, BnTT8-2 and BnTT8-3 have very high homology at amino acid level, BnTT8-1 and BnTT8-2 share 96.7% identities and 98.1% positives, BnTT8-1 and BnTT8-3 share 97% identities and 98.6% positives, while BnTT8-2 and BnTT8-3 share 98.4% identities and 99.3% positives. The nucleotide sequence and the protein sequence of BnTT8 genes shared high homologies with those of the known plant's TT8 genes and the bHLH genes, especially with the highest homologies to those of the AtTT8 gene. The BnTT8-1, BnTT8-2 and BnTT8-3 share 83.4%, 83.9% and 83.9% of similaritis and 76.5%, 76.9% and 77.5% identities with AtTT8, respectively.
BnTT8-1, BnTT8-2 and BnTT8-3 all have a bHLH conserved domain from L_(366) to H_(417). At this region and other conserved residues, they are extremely similar to AtTT8. BnTT8-1, BnTT8-2 and BnTT8-3 had 33, 33 and 32 potential phosphorylation sites respectively, suggesting that phosphorylation might be related to the activity of the BnTT8 proteins. The secondary structures of the BnTT8 proteins are primarily composed of alpha helix and random coil, except for certain proportions of extended strand and beta turn.
Like AtTT8, the 3 BnTT8 members is probably involved in regulation of flavonoid structural genes. But since BnTT8-3 lacks the 86-bp C-terminal region, whether it is bioactive and how active it is deserve further study.
2. Number of the members of BnTT8 gene family
In Southern blot results, each restriction enzyme yielded 3 or less hybridization bands. In this study, only 3 members were cloned. Thus it is postulated that there are only 3 members of BnTT8 gene famliy in B. napus.
3. Construction of a BnTT8 antisense plant expression vector
A 817-bp fragment conserved in BnTT8 gene family was integrated into the intemdiate expression vector pCambia2301G in the anti orientation to replace the GUS gene driven by the CaMV 35S promoter. Thus a BnTT8 antisense plant expression vector was constructed. The vector was named as pFBnTT8A..
4. Obtaining of the antisense-BnTT8 transgenic pantlets
The transformation of a B. napus cultivar Xiangyou 15 mediated by Agrobacterium tumefaciens carrying the BnTT8 antisense expression vector pFBnTT8A was made. 24 plantlets with kanamycin resistance were obtained, among which 13 plantlets were identified as transgenic plants by detecting the expression of the GUS reporter gene. The transformation effciency was 2.23%.
拟南芥TT8(AtTT8)是类黄酮合成途径中的一个调控因子，它属于碱性螺旋-环-螺旋(basic-Helix-loop-helix domain，bHLH)转录因子，此类转录因子在生物的发育过程中具有重要作用，拟南芥、玉米、矮牵牛、金鱼草等植物中该基因的突变导致种子颜色或花色改变。拟南芥中，TT8、TTG1、TT2三个蛋白都调控BAN基因和DFR基因的转录。研究油菜TT8基因有助于进一步了解甘蓝型油菜黄籽性状形成的分子机制，可以为通过基因工程创造稳定的黄籽甘蓝型油菜新材料提供基础。
本研究克隆了甘蓝型油菜TT8基因家族的3个成员的全长cDNA和1个成员的基因组序列，并对其序列及编码蛋白进行了系统的分析；通过Sourthern杂交鉴定了TT8基因家族在甘蓝型油菜中的成员数；构建了甘蓝型油菜TT8基因家族反义植物表达载体，并转化黑籽甘蓝型油菜得到了转基因植株。主要结果如下：
1、甘蓝型油菜BnTT8基因家族3个成员的克隆和分析
根据AtTT8保守区设计引物，以甘蓝型油菜黑籽系5B生殖器官总RNA为材料，利用RACE(rapid amplification of cDNA ends)技术，克隆了BnTT8基因家族的3个成员的全长cDNA序列，分别命名为BnTT8-1、BnTT8-2、BnTT8-3，并克隆了BnTT8-1的基因组序列。
BnTT8-1基因组序列长2949bp，含6个内含子，mRNA(不包括polyA，下同)为1771bp，开放阅读框(ORF，包括终止密码子，下同)为1566bp，5’UTR为147bp，3’UTR为58bp。BnTT8-2mRNA为1783bp，ORF为1566bp，5’UTR为137bp，3’UTR为80bp。BnTT8-3 mRNA为1676bp，的ORF为1308bp，5’UTR为134bp，3’UTR为231bp。在BnTT8-1、BnTT8-2和BnTT8-3在3’UTR均存在典型的AATAAA加尾信号，在5’UTR区都有富含AG重复的微卫星序列。核苷酸组成在不同区段变化较大，非编码区G+C含量明显低于编码区。BnTT8-1的内含子的总长度比拟南芥大大缩短。
推导的BnTT8-1蛋白为521个氨基酸，Mw=59.669kDa，pI=5.61；BnTT8-2为1个521个氨基酸，Mw=59.955kDa，pI=5.65；BnTT8-3为435个氨基酸，Mw=50.071kDa，pI=5.47。3个蛋白中均以谷氨酸的含量为最高，且酸性氨基酸数目多于碱性的氨基酸。BnTT8-3 mRNA对应于其它TT8基因的终止密码子发生突变，且与其它TT8 mRNA相比在该位置上游发生了一段107bp的序列缺失，导致编码的蛋白比其它TT8蛋白在C-末端缺少86个氨基酸。
BnTT8-1、BnTT8-2和BnTT8-3之间在氨基酸水平上有很高的同源性，BnTT8-1和BnTT8-2的一致性和相似性分别为96.7％和98.1％，BnTT8-1和BnTT8-3的一致性和相似性分别为97％和98.6％，BnTT8-2和BnTT8-3的一致性和相似性分别为98.4％和99.3％。BnTT8基因的核酸及蛋白序列与已知植物的TT8基因和bHLH基因的核酸及蛋白序列有高度的同源性，尤与AtTT8最相似。BnTT8-1与AtTT8的相似性为83.4％，一致性为76.5％；BnTT8-2与AtTT8的相似性为83.9％，一致性为76.9％；BnTT8-3与AtTT8的相似性为83.9％，一致性为77.5％。BnTT8-1、BnTT8-2和BnTT8-3蛋白都在L_(366)到H_(417)区域分别对应存在HLH保守性结构域，在该结构域及保守性氨基酸位置，它们与AtTT8高度相似。它们分别有33个、33个和32个潜在的磷酸化位点，说明磷酸化作用可能与它们的活性有关。它们的二级结构主要由a螺旋和随机卷曲组成，延伸链和β转角也占一定比例。
BnTT8家族3个成员可能和AtTT8一样，调控类黄酮途径的结构基因表达。由于BnTT8-3蛋白的C-末端缺失了86个氨基酸，所以该成员蛋白是否具有活性，活性强弱如何，值得进一步研究。
2、甘蓝型油菜BnTT8基因家族的成员数
Southern blot杂交结果中，每种限制酶均获得3条或3条以下的杂交条带。而本研究采用竭尽式RACE克隆方式，也只获得了3个成员的全长cDNA。因此推测，甘蓝型油菜中BnTT8基因家族可能只存在3个成员。
3、BnTT8反义表达载体的构建
将BnTT8基因家族共保守的817bp的片段反接到中间表达载体pCambia2301G中，替换其上的GUS基因，形成CaMV 35S启动子驱动、反义共抑制所有BnTT8成员的植物表达载体，命名为pFBnTT8A。
4、甘蓝型油菜TT8反义转化植株的获得
采用农杆菌介导法转化甘蓝型油菜“湘油15”，获得了24株抗卡那霉素的再生苗，通过检测GUS基因在抗性植株中的稳定表达情况，发现其中13株为稳定表达的转基因植株，转化频率达到2.23％。
Oilseed rape (Brassica napus L.) is one of the four major oil crops in the world and is one of the most important oil plants in China too. In recent year the yellow-seeded B. napus has attracted researchers' interest in that it has many good properties compared to black seeds, such as thinner seed coat, fewer seed coat pigment, lower meal fiber content, higher seed oil content and meal protein content, etc. It is one of the most important objectives to breed yellow-seeded rapeseed cultivars with stably inherited yellow seed trait and good agronomic traits. The molecular mechanism of the yellow seed trait is still unclear in B. napus. However, the achievements on the Transparent Testa (TT) trait in Arabidopsis thaliana are available for an insight into the formation of the yellow seed color in B. napus. Up to now, 21 TT loci and a BAN locus have been identified, and many of the TT genes have been cloned in A thaliana.
The A. thaliana TT8 (AtTT8) gene encodes a regulative factor in the flavanone synthesis way. The AtTT8 belongs to the basic helix-loop-helix domain (bHLH) transcription factors which play an important role in the biologic development. In A thaliana, Zea mays, Petunia hybrida, Antirrhinum majus and so on, the TT8 (or analogue) gene mutation alters seed color or flower color. In A. thaliana, the TT8, TTG1 and TT2 proteins all regulate the BAN and DFR genes' transcription. Study on B. napus TT8 gene is helpful to understand the molecular mechanism of the yellow seed color formation and may lay a foundation for creating stable yellow-seeded lines by genetic engineering.
In this research, full-length cDNAs of 3 members of B. napus TT8 gene family (BnTT8) were isolated, together with genomic sequence of one member. The nucleotide sequences and the corresponding polypeptides of the BnTT8 gene family were systematically analyzed. The mumber of the BnTT8 gene family members was identified through Sourthern-blot. A BnTT8 gene family antisense plant expression vector was constructed and some regenerated plantlets were obtained from black-seeded B. napus explant cultivar transformed with this vector.
1. Cloning and analysis of the 3 members of BnTT8 gene family in B. napus
Based on the conserved regions of the A. thaliana TT8 gene, the PCR pimers were designed. 3 full-length of cDNA sequences of the B. napus TT8 gene famliy were cloned using total RNA from reproductive organs of black-seeded B.napus line 5B by RACE (rapid amplification of cDNA ends) technique. They were named as BnTT8-1, BnTT8-2 and BnTT8-3, respectively. Furthermore, the genomic sequence of BnTT8-1 was also cloned.
BnTT8-1 has a genomic sequence of 2 949bp with 6 introns. Its mRNA (excluding the polyA tail) is 1 771 bp, with an open reading frame (ORF, including the stop codon) of 1 566bp. Its 5' UTR and 3' UTR are 147bp and 58bp respectively. BnTT8-2 mRNA is 1 783bp, with an ORF of 1 566bp, a 5' UTR of 137bp, and a 3' UTR of 80bp. BnTT8-3 mRNA is 1 676bp, with an ORF of 1 308bp, a 5' UTR of 137bp and a 3' UTR of 231bp. There is a typical polyadenylation signal AATTAAA in 3' UTR of them, and there are microsattilites rich in AG-repeats in their 5' UTRs. The nucleotide composition is varied in different regions of them. The G+C content of the non-coding region is obviously lower than that of the coding region. In BnTT8-1, the total intron length is much shorter than A. thaliana.
The deduced BnTT8-l protien is 521 aa, Mw=59.669 kDa, pI=5.61; BnTT8-2 is also 521 aa, Mw=59.955 kDa, pI=5.65; and BnTT8-3 is 435 aa, Mw=50.071 kDa, pI=5.47. Glutamic acid is the most abundant residue in them, and acidic residues are more than basic ones. BnTT8-3 mRNA shows mutation at position corresponding stop codon of typical TT8 genes, and a 107-bp deletion exists just upstream this site, causing the loss a 86-aa C-terminal region in its deduced protein.
BnTT8-1, BnTT8-2 and BnTT8-3 have very high homology at amino acid level, BnTT8-1 and BnTT8-2 share 96.7% identities and 98.1% positives, BnTT8-1 and BnTT8-3 share 97% identities and 98.6% positives, while BnTT8-2 and BnTT8-3 share 98.4% identities and 99.3% positives. The nucleotide sequence and the protein sequence of BnTT8 genes shared high homologies with those of the known plant's TT8 genes and the bHLH genes, especially with the highest homologies to those of the AtTT8 gene. The BnTT8-1, BnTT8-2 and BnTT8-3 share 83.4%, 83.9% and 83.9% of similaritis and 76.5%, 76.9% and 77.5% identities with AtTT8, respectively.
BnTT8-1, BnTT8-2 and BnTT8-3 all have a bHLH conserved domain from L_(366) to H_(417). At this region and other conserved residues, they are extremely similar to AtTT8. BnTT8-1, BnTT8-2 and BnTT8-3 had 33, 33 and 32 potential phosphorylation sites respectively, suggesting that phosphorylation might be related to the activity of the BnTT8 proteins. The secondary structures of the BnTT8 proteins are primarily composed of alpha helix and random coil, except for certain proportions of extended strand and beta turn.
Like AtTT8, the 3 BnTT8 members is probably involved in regulation of flavonoid structural genes. But since BnTT8-3 lacks the 86-bp C-terminal region, whether it is bioactive and how active it is deserve further study.
2. Number of the members of BnTT8 gene family
In Southern blot results, each restriction enzyme yielded 3 or less hybridization bands. In this study, only 3 members were cloned. Thus it is postulated that there are only 3 members of BnTT8 gene famliy in B. napus.
3. Construction of a BnTT8 antisense plant expression vector
A 817-bp fragment conserved in BnTT8 gene family was integrated into the intemdiate expression vector pCambia2301G in the anti orientation to replace the GUS gene driven by the CaMV 35S promoter. Thus a BnTT8 antisense plant expression vector was constructed. The vector was named as pFBnTT8A..
4. Obtaining of the antisense-BnTT8 transgenic pantlets
The transformation of a B. napus cultivar Xiangyou 15 mediated by Agrobacterium tumefaciens carrying the BnTT8 antisense expression vector pFBnTT8A was made. 24 plantlets with kanamycin resistance were obtained, among which 13 plantlets were identified as transgenic plants by detecting the expression of the GUS reporter gene. The transformation effciency was 2.23%.
引文
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