茶树咖啡酸氧甲基转移酶基因的克隆及表达分析
摘要
茶树(Camellia sinensis)是重要的木本经济作物之一。木质素是植物界中仅次于纤维素的天然有机资源,茶树体内木质素的含量和各单体比例与茶树抗逆性、茶叶品质等方面有关。咖啡酸氧甲基转移酶(caffeic acid o-methyltransferase,COMT)是木质素生物合成中的关键酶之一。本研究以元宵绿品种为材料,克隆了COMT基因cDNA全长,并进行了原核表达及实时定量分析,以期为将来通过调控该基因的表达量,提高茶树叶片的持嫩性及茶树的抗逆性奠定基础。主要研究结果如下:
(1)根据其他作物中已克隆的COMT基因氨基酸序列,设计简并引物,通过RT-PCR扩增得到317 bp的特异片段,通过RACE法获得茶树COMT基因cDNA,全长为1381 bp,Genbank登录号为HM204933。此基因包含一个长1092 bp的开放阅读框,编码393个氨基酸,5’端非编码区和3’端非编码区分别为77 bp、212 bp。经GenBank数据库Blast分析,其核苷酸序列与欧洲白桦、苎麻、中果咖啡和毛果杨的COMT基因相似性分别为80%、78%、77%、77%,氨基酸序列与毛果杨的COMT基因相似性达到100%。
(2)设计特异引物,PCR扩增COMT开放阅读框片段,插入到表达载体pET-32a中,转化表达宿主菌BL21trxB(DE3),IPTG诱导后高效表达产生分子量约为60 KD的融合蛋白。该融合蛋白包括20 kD的Trx(硫氧还蛋白)和40 kD的COMT蛋白。随着诱导时间的变化,融合蛋白表达量递增,表明COMT基因在大肠杆菌中得到了表达。
(3)实时荧光定量PCR结果表明,茶树不同生长阶段的叶片中都有COMT基因的表达,其表达量随着成熟度的增大而增大。
(4)通过对水古、浙江紫笋、龙井43、平阳特早四个品种的芽下第一叶中COMT基因的表达量分析得到,其表达量大小依次为龙井43>水古>浙江紫笋>平阳特早,其结果与材料采摘时的成熟程度一致。
Tea (Camellia sinensis) is one of the important tree crops. Tea is the world's most popular non-alcoholic beverage, it is good for health with physiological function. Lignin is a kind of important macromolecular organic material and only inferior to cellulose inside plant.The content and composition of lignin in tea are related with treatment -resistant, tea quality etc. Caffeic acid o-methyltransferase (COMT) is one of the key enzymes in the lignin synthetic system. We cloned the COMT gene from the Yuan Xiao lv , analyze by protokaryon expression and real-tme PCR.We hope the results will be helpful to improve the reatment–resistant and tea quality.The main results are follows,
(1) Through the blasting between public sequences in other plants obtained fragment 317 bp. Through RACE obtained full length sequence of COMT cDNA which coding 393 amino acid was 1381bp,including 1092bp ORF,77 bp 5’UTR and 212 bp 3’UTR.The accession number is HM204933.The cDNA sequence shows a significant homology to the COMT genes from Betula pendula (80 %),Boehmeria nivea (78 %) ,Coffea canephora (77 %) ,Populus trichocarpa(77 %).The amino acid sequences share 100% identity with Populus trichocarpa.
(2) By PCR obtained the ORF of COMT gene,recombined plasmid pET-32 and the ORF of COMT,then transformed into BL21 trxB (DE3).The result displayed a special protein which molecular was 60 KD was produced by IPTG induced.The production including Trx which molecular was 20KD coupling fusion protein and 40 KD protein that was encoded by COMT.The expression of the reconstructing protein was increased with the induced time.
(3) The result of tissue expression distribution of COMT gene in tea Ping Yang Te Zao variety is that expression of the reconstructing protein was increased with the grown age.
(4) The result of expression of COMT gene in the first leave between varieties is Longjin43> Shuigu > Zhe Jiang Zi Sun >Ping Yang Te Zao.
(1)根据其他作物中已克隆的COMT基因氨基酸序列,设计简并引物,通过RT-PCR扩增得到317 bp的特异片段,通过RACE法获得茶树COMT基因cDNA,全长为1381 bp,Genbank登录号为HM204933。此基因包含一个长1092 bp的开放阅读框,编码393个氨基酸,5’端非编码区和3’端非编码区分别为77 bp、212 bp。经GenBank数据库Blast分析,其核苷酸序列与欧洲白桦、苎麻、中果咖啡和毛果杨的COMT基因相似性分别为80%、78%、77%、77%,氨基酸序列与毛果杨的COMT基因相似性达到100%。
(2)设计特异引物,PCR扩增COMT开放阅读框片段,插入到表达载体pET-32a中,转化表达宿主菌BL21trxB(DE3),IPTG诱导后高效表达产生分子量约为60 KD的融合蛋白。该融合蛋白包括20 kD的Trx(硫氧还蛋白)和40 kD的COMT蛋白。随着诱导时间的变化,融合蛋白表达量递增,表明COMT基因在大肠杆菌中得到了表达。
(3)实时荧光定量PCR结果表明,茶树不同生长阶段的叶片中都有COMT基因的表达,其表达量随着成熟度的增大而增大。
(4)通过对水古、浙江紫笋、龙井43、平阳特早四个品种的芽下第一叶中COMT基因的表达量分析得到,其表达量大小依次为龙井43>水古>浙江紫笋>平阳特早,其结果与材料采摘时的成熟程度一致。
Tea (Camellia sinensis) is one of the important tree crops. Tea is the world's most popular non-alcoholic beverage, it is good for health with physiological function. Lignin is a kind of important macromolecular organic material and only inferior to cellulose inside plant.The content and composition of lignin in tea are related with treatment -resistant, tea quality etc. Caffeic acid o-methyltransferase (COMT) is one of the key enzymes in the lignin synthetic system. We cloned the COMT gene from the Yuan Xiao lv , analyze by protokaryon expression and real-tme PCR.We hope the results will be helpful to improve the reatment–resistant and tea quality.The main results are follows,
(1) Through the blasting between public sequences in other plants obtained fragment 317 bp. Through RACE obtained full length sequence of COMT cDNA which coding 393 amino acid was 1381bp,including 1092bp ORF,77 bp 5’UTR and 212 bp 3’UTR.The accession number is HM204933.The cDNA sequence shows a significant homology to the COMT genes from Betula pendula (80 %),Boehmeria nivea (78 %) ,Coffea canephora (77 %) ,Populus trichocarpa(77 %).The amino acid sequences share 100% identity with Populus trichocarpa.
(2) By PCR obtained the ORF of COMT gene,recombined plasmid pET-32 and the ORF of COMT,then transformed into BL21 trxB (DE3).The result displayed a special protein which molecular was 60 KD was produced by IPTG induced.The production including Trx which molecular was 20KD coupling fusion protein and 40 KD protein that was encoded by COMT.The expression of the reconstructing protein was increased with the induced time.
(3) The result of tissue expression distribution of COMT gene in tea Ping Yang Te Zao variety is that expression of the reconstructing protein was increased with the grown age.
(4) The result of expression of COMT gene in the first leave between varieties is Longjin43> Shuigu > Zhe Jiang Zi Sun >Ping Yang Te Zao.
引文
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