TMV-RH基因组近全长序列测定及CP基因和MP基因对烟草的遗传转化
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摘要
根据已发表的TMV基因组的核苷酸序列设计5对简并引物，以感染TMV的地黄（Rehmannia glutinosa）叶片总RNA为模板，经RT-PCR扩增获得TMV-RH的5个核苷酸片段的cDNA。分别将其克隆到pMD-18-T vector上，得到5个含有目的cDNA片断的重组质粒。对重组质粒的测序结果表明，这5个片段相互重叠并覆盖TMV全基因组，经拼接后获得了TMV-RH的近全长基因组序列。根据已测得的6000bp核苷酸序列与TMV-U1株系的比较结果可知，TMV-RH基因组含有4个开放阅读框（ORF），分别编码复制酶（126KD/183KD蛋白）基因、移动蛋白（MP，30KD蛋白）基因和外壳蛋白（CP，17.5KD蛋白）基因，还有两端的两个非编码区。利用DNAMAN对所测序列与TMV-U1株系的核苷酸序列进行了比较，结果表明，所测序列包含三个完整的基因：TMV-RH MP基因全长为804nt，比TMV-U1株系缺少3个碱基，起始密码子为AUG，终止密码子为UAA，推测编码267个氨基酸，与TMV-U1株系的核苷酸序列和由此推导的氨基酸序列同源性分别为81.2%和84.6%； CP基因 ORF长度为480nt，起始密码子为AUG，终止密码子为UGA，推测编码159个氨基酸，与TMV U1株系CP基因的核苷酸同源性为86.5%，氨基酸同源性为94.3%；54KD蛋白的基因包括1503nt，起始密码子为AUG，终止密码子为UAA，推测编码499个氨基酸，与TMV U1株系的核苷酸同源性为85.6%，氨基酸同源性为95.8%。利用BLAST对CP、MP和54KD进行的分析表明，我们鉴定的TMV-RH MP基因的核苷酸序列与已发表的TMV其它株系MP基因的核苷酸序列的同源性在71.0%-81.2%之间，氨基酸序列同源性在72.6%-83.9%之间，与番茄花叶病毒（ToMV）MP基因的核苷酸同源性为71.0%，氨基酸序列同源性为72.6%；CP基因的核苷酸序列与已发表的TMV其它株系CP基因的核苷酸序列的同源性在
76.3%-88.5%之间，氨基酸序列同源性在79.3%-95.0%之间，与番茄花叶病毒CP基因的核苷酸同源性为77.1%，氨基酸序列同源性为81.1%；54KD基因的核苷酸序列与已发表的TMV其它株系的核苷酸序列的同源性在81.6%-85.5%之间，氨基酸序列同源性在92.0%-100%之间，与番茄花叶病毒的核苷酸同源性为81.9%，氨基酸序列同源性为95.8%。不同株系MP、CP和54KD基因的核甘酸序列和氨基酸序列进化树分析结果也表明，TMV-RH可明显地划分为一个类型。由此我们推测TMV-RH可能为烟草花叶病毒的一个新株系。目前我们正在利用RACE法对TMV-RH基因组的5’端和3’端序列进行测定。
本文还针对TMV-RH 的CP基因设计引物，以感染TMV-RH的地黄叶片总RNA为模板，经RT-PCR扩增获得TMV-RH CP基因的cDNA片段。将CP基因的cDNA片段连接到T载体上，得到含有完整CP基因的重组质粒pTCP。然后将CP基因通过中间载体 pJIT163克隆到双元植物表达载体pBinplus上，构建成功CP基因的植物表达载体pJBCP。利用CP基因的植物表达载体和我们以前获得的全长和缺失的MP基因的植物表达载体（pBMP-F和pBMP-D）在土壤农杆菌LBA4404（Rif＋）介导下，以叶盘法分别转化本氏烟。经共培养和分化选择培养，共获得了40株再生植株。经PCR鉴定，其中36株能扩增出特异性目的片段，说明初步获得了转基因烟草植株。目前正在对烟草再生植株进行Southern杂交和Northern杂交以及抗病性鉴定。
According to the published genomic sequence of TMV ,five sets of PCR primers were designed and five nucleotide segments were amplified by RT-PCR from the total RNA of the TMV infected Rehmannia glutinosa leaves. The five PCR products were cloned into pMD-18-T vector and the recombinant plasmids were obtained. The sequences of the cloned PCR products were analysed and the results show that the five segments cover the complete genomes of TMV-RH isolates. The five nucleotide segments were linked and the genomic sequence of TMV-RH were determined .The analyse of the sequence show that TMV-RH comprises four ORFs , including replicase gene,MP gene and CP gene, and two non-translation-regions(UTR). The DNAMAN analyse show, MP gene contains 804nt, lack of 3nt than that of U1, the start-code is UAG and end-code UAA, it encodes 499 amino acids(aa) and its theoretical molecular weight is 54KD, the nucleotide sequence identity and the deduced amino acid sequence identity with that of TMV-U1 were 81.2% and 84.6%; CP gene contains 480nt, the start-code its ORF is AUG and end-code UGA, it encodes 159 amino acids(aa) and its theoretical molecular weight is 17.5KD, the nucleotide sequence identity and the deduced amino acid sequence identity with that of TMV-U1 were 86.5% and 94.3 %; 54KD gene contains 1503nt, the start-code is AUG and end-code UAA, it encodes 267 amino acids(aa) and its theoretical molecular weight is 30KD, the nucleotide sequence identity and the deduced amino acid sequence identity with that of TMV-U1 were 85.6% and 95.8%.The BLAST results show that the nucleotide and aa sequence of MP were 71.0% to 81.2% and 72.6% to 84.6% identical to other tobamoviruses respectively ; the CP were 76.3% to 88.5% and 79.3 to 95.0% ; the 54KD were 81.6% to 85.5% and 92.0% to 100% . Based on the nucleotide sequences of MP and CP genes ,and 54KD gene, the comparison of the TMV-RH isolate with other TMV isolates confirm the TMV-RH is a new strain of
TMV. The identification work of its 3’-end and 5’-end are still in processing now.
Furthermore, this article had cloned the CP gene . it were cloned into the binary vector pBinplus with the help of the intermidiate vector pJIT163 to obtain the plant expression vector pJBCP. pJBCP carrying CP gene and pBMP-F、pBMP-D carrying full MP and deleted MP gene respectively were used for transformation to tobacco by LBA4404. Transgentic tobacco have obtained through leaf discs transformation and were identified by PCR. We have obtained 36 transgenetic tabacco plants out of 40. Southern blot, Northern blot test and the resistance test are still in processing.
76.3%-88.5%之间，氨基酸序列同源性在79.3%-95.0%之间，与番茄花叶病毒CP基因的核苷酸同源性为77.1%，氨基酸序列同源性为81.1%；54KD基因的核苷酸序列与已发表的TMV其它株系的核苷酸序列的同源性在81.6%-85.5%之间，氨基酸序列同源性在92.0%-100%之间，与番茄花叶病毒的核苷酸同源性为81.9%，氨基酸序列同源性为95.8%。不同株系MP、CP和54KD基因的核甘酸序列和氨基酸序列进化树分析结果也表明，TMV-RH可明显地划分为一个类型。由此我们推测TMV-RH可能为烟草花叶病毒的一个新株系。目前我们正在利用RACE法对TMV-RH基因组的5’端和3’端序列进行测定。
本文还针对TMV-RH 的CP基因设计引物，以感染TMV-RH的地黄叶片总RNA为模板，经RT-PCR扩增获得TMV-RH CP基因的cDNA片段。将CP基因的cDNA片段连接到T载体上，得到含有完整CP基因的重组质粒pTCP。然后将CP基因通过中间载体 pJIT163克隆到双元植物表达载体pBinplus上，构建成功CP基因的植物表达载体pJBCP。利用CP基因的植物表达载体和我们以前获得的全长和缺失的MP基因的植物表达载体（pBMP-F和pBMP-D）在土壤农杆菌LBA4404（Rif＋）介导下，以叶盘法分别转化本氏烟。经共培养和分化选择培养，共获得了40株再生植株。经PCR鉴定，其中36株能扩增出特异性目的片段，说明初步获得了转基因烟草植株。目前正在对烟草再生植株进行Southern杂交和Northern杂交以及抗病性鉴定。
According to the published genomic sequence of TMV ,five sets of PCR primers were designed and five nucleotide segments were amplified by RT-PCR from the total RNA of the TMV infected Rehmannia glutinosa leaves. The five PCR products were cloned into pMD-18-T vector and the recombinant plasmids were obtained. The sequences of the cloned PCR products were analysed and the results show that the five segments cover the complete genomes of TMV-RH isolates. The five nucleotide segments were linked and the genomic sequence of TMV-RH were determined .The analyse of the sequence show that TMV-RH comprises four ORFs , including replicase gene,MP gene and CP gene, and two non-translation-regions(UTR). The DNAMAN analyse show, MP gene contains 804nt, lack of 3nt than that of U1, the start-code is UAG and end-code UAA, it encodes 499 amino acids(aa) and its theoretical molecular weight is 54KD, the nucleotide sequence identity and the deduced amino acid sequence identity with that of TMV-U1 were 81.2% and 84.6%; CP gene contains 480nt, the start-code its ORF is AUG and end-code UGA, it encodes 159 amino acids(aa) and its theoretical molecular weight is 17.5KD, the nucleotide sequence identity and the deduced amino acid sequence identity with that of TMV-U1 were 86.5% and 94.3 %; 54KD gene contains 1503nt, the start-code is AUG and end-code UAA, it encodes 267 amino acids(aa) and its theoretical molecular weight is 30KD, the nucleotide sequence identity and the deduced amino acid sequence identity with that of TMV-U1 were 85.6% and 95.8%.The BLAST results show that the nucleotide and aa sequence of MP were 71.0% to 81.2% and 72.6% to 84.6% identical to other tobamoviruses respectively ; the CP were 76.3% to 88.5% and 79.3 to 95.0% ; the 54KD were 81.6% to 85.5% and 92.0% to 100% . Based on the nucleotide sequences of MP and CP genes ,and 54KD gene, the comparison of the TMV-RH isolate with other TMV isolates confirm the TMV-RH is a new strain of
TMV. The identification work of its 3’-end and 5’-end are still in processing now.
Furthermore, this article had cloned the CP gene . it were cloned into the binary vector pBinplus with the help of the intermidiate vector pJIT163 to obtain the plant expression vector pJBCP. pJBCP carrying CP gene and pBMP-F、pBMP-D carrying full MP and deleted MP gene respectively were used for transformation to tobacco by LBA4404. Transgentic tobacco have obtained through leaf discs transformation and were identified by PCR. We have obtained 36 transgenetic tabacco plants out of 40. Southern blot, Northern blot test and the resistance test are still in processing.
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