红花bHLH转录因子基因的克隆、表达分析及植物表达载体构建
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摘要
克隆红花花瓣中bHLH(basic helix-loop-helix)基因,研究其在红花不同开花时期花瓣中的表达量并构建其植物表达载体。利用红花基因组测序结果中富集到的bHLH家族中的基因CtbHLH1的开放阅读框,设计特异性引物进行PCR扩增。利用RT-PCR法分析在红花不同开花时期花瓣中bHLH1基因的表达量,同时构建植物表达载体p BASTA-bHLH1。获得bHLH1基因全长897 bp,编码298个氨基酸。红花bHLH1与其他物种氨基酸具有一定的同源性,其与烟草的氨基酸序列相似性最高。实时荧光定量PCR分析表明,CtbHLH1基因在红花不同组织及不同开花时期的花瓣中表达水平具有显著差异,其在花瓣中表达量高,开花第3天表达量最高,末花期表达量低。另外其在根中有表达,在茎、叶中表达量极低。该研究成功地对bHLH1基因进行克隆及表达分析,并构建植物表达载体p BASTA-bHLH1。
To clone bHLH( basic helix-loop-helix) gene from Carthamus tinctorius,analyze the expression level in different plant tissues and construct the plant expression vector. The bHLH1 gene was cloned by RT-PCR techniques,and the protein characteristics were analyzed by bioinformatics,and phylogenetic tree was constructed. The expression of bHLH1 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR,and the plant expression vector p BASTA-bHLH1 was constructed. The obtained ORF sequence of bHLH1 gene was 897 bp,encoded a protein of 298 amino acids. Sequence alignment and phylogenetic tree analyses showed that C. tinctorius bHLH1 had a certain homology with other species of amino acids,and was the most similar to the amino acid sequence of tobacco. Real-time PCR results showed significant differences,CtbHLH1 gene in red flower petals in different tissues and different flowering period had remarkable difference in expression level,its high amount expressed in petals,flowers third day after blossom expressed the highest quantity,at the end of the flowering the expression quantity is low. In addition,it is expressed in the root,and the expression in the stem and leaves is extremely low. The bHLH1 gene of C. tinctorius is successfully cloned,and the expression is analyzed. The plant expression vector p BASTA-bHLH is constructed.
To clone bHLH( basic helix-loop-helix) gene from Carthamus tinctorius,analyze the expression level in different plant tissues and construct the plant expression vector. The bHLH1 gene was cloned by RT-PCR techniques,and the protein characteristics were analyzed by bioinformatics,and phylogenetic tree was constructed. The expression of bHLH1 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR,and the plant expression vector p BASTA-bHLH1 was constructed. The obtained ORF sequence of bHLH1 gene was 897 bp,encoded a protein of 298 amino acids. Sequence alignment and phylogenetic tree analyses showed that C. tinctorius bHLH1 had a certain homology with other species of amino acids,and was the most similar to the amino acid sequence of tobacco. Real-time PCR results showed significant differences,CtbHLH1 gene in red flower petals in different tissues and different flowering period had remarkable difference in expression level,its high amount expressed in petals,flowers third day after blossom expressed the highest quantity,at the end of the flowering the expression quantity is low. In addition,it is expressed in the root,and the expression in the stem and leaves is extremely low. The bHLH1 gene of C. tinctorius is successfully cloned,and the expression is analyzed. The plant expression vector p BASTA-bHLH is constructed.
引文
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[12] Li X,Chen L,Hong M,et al. A large insertion in bHLH transcription factor BrTT8 resulting in yellow seed coat in Brassica rapa[J]. PLoS ONE,2012,7(9):e44145.
[13]吕娟娟,王进军,高新菊,等.二斑叶螨乙酰辅酶A羧化酶基因全长c DNA克隆及其生物信息学分析[J].中国农业科学,2013,46(8):1736.
[14] Albert N W,Dames K M,Lewis D H,et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots[J]. Plant Cell,2014,26(3):962.
[15]涂华,陈碧琼,张燕军.天然类黄酮物质的提取工艺研究进展[J].中国实验方剂学杂志,2011,17(6):277.
[16]顾超兰,周杰.红花黄色素对佐剂型关节炎大鼠的抗炎作用研究[J].中国现代应用药学,2017,34(4):521.
[17] Liu X F,Yin X R,Allan A C,et al. The role of MrbHLH1 and Mr MYB1 in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry(Myrica rubra)during anthocyanin biosynthesis[J]. Plant Cell Tiss Org Cult,2013,115(3):285.
[18]刘秀明,杨文婷,张雪萌,等.红花查尔酮异构酶基因全长c DNA的克隆及表达分析[J].西北农林科技大学学报:自然科学版,2015,43(7):201.
[2] Zhou X,Tang L,Xu Y,et al. Towards a better understanding of medicinal uses of Carthamus tinctorius L. in traditional Chinese medicine:a phytochemical and pharmacological review[J].J Ethnopharmacol,2014,151(1):27.
[3] DuanJ L,Wang J W,Guan Y,et al. Safflor yellow A protects neonatal rat cardiomyocytes against anoxia/reoxygenation injury in vitro[J]. Acta Pharmacol Sin,2013,34(4):487.
[4] Han S Y,Li H X,Ma X,et al. Protective effects ofpurified safflower extract on myocardial ischemia in vivo and in vitro[J].Phytomedicine,2009,16:694.
[5] Wang C,He Y,Yang M,et al. Safflor yellow B suppresses angiotensinⅡ-mediated human umbilical vein cell injury via regulation of Bcl-2/p22(phox)expression[J]. Toxicol Appl Pharmacol,2013,273(1):59.
[6] Li J,Li Y M,Qiao B Y,et al. The value of safflower yellow injection for the treatment of acute cerebral infarction:a randomized controlled trial[J]. Evid Based Complement Alternat Med,2015,2015:478793.
[7] Fan S,Lin N,Shan G,et al. Safflower yellow for acute ischemic stroke:a systematic review of randomized controlled trials[J].Complement Ther Med,2014,22(2):354.
[8]吴铁松,吴丽霞,杨慧文.红花总黄素对糖尿病大鼠血管内皮功能的保护作用[J].今日药学,2013,23(4):220.
[9] SunH,Fan H J,Ling H Q. Genome-wide identification and characterization of the b HLH gene family in tomato[J]. BMC Genomics,2015,16(1):9.
[10] Ludwing S R,Habera L F,Dellaporta S L,et al. Lc,a member of the maize R gene family responsible for tissue-specific anthocyanin production,encodes a protein similar to transcriptional activators and contains the myc-homology region[J]. Proc Natl Acad Sci USA,1989,86:7092.
[11] Baudry A,Caboche M,Lepiniec L. TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors,allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana[J]. Plant J,2006,46:768.
[12] Li X,Chen L,Hong M,et al. A large insertion in bHLH transcription factor BrTT8 resulting in yellow seed coat in Brassica rapa[J]. PLoS ONE,2012,7(9):e44145.
[13]吕娟娟,王进军,高新菊,等.二斑叶螨乙酰辅酶A羧化酶基因全长c DNA克隆及其生物信息学分析[J].中国农业科学,2013,46(8):1736.
[14] Albert N W,Dames K M,Lewis D H,et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots[J]. Plant Cell,2014,26(3):962.
[15]涂华,陈碧琼,张燕军.天然类黄酮物质的提取工艺研究进展[J].中国实验方剂学杂志,2011,17(6):277.
[16]顾超兰,周杰.红花黄色素对佐剂型关节炎大鼠的抗炎作用研究[J].中国现代应用药学,2017,34(4):521.
[17] Liu X F,Yin X R,Allan A C,et al. The role of MrbHLH1 and Mr MYB1 in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry(Myrica rubra)during anthocyanin biosynthesis[J]. Plant Cell Tiss Org Cult,2013,115(3):285.
[18]刘秀明,杨文婷,张雪萌,等.红花查尔酮异构酶基因全长c DNA的克隆及表达分析[J].西北农林科技大学学报:自然科学版,2015,43(7):201.
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