橡胶树HbCPA基因的克隆、表达及生物信息学分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0294

摘要/Abstract

摘要：

N-氨甲酰基腐胺酰胺水解酶（N-carbamoylputreseine amidohydrolase,CPA）是高等植物通过精氨酸途径合成腐胺的终反应酶。利用RACE技术从橡胶树中克隆了一个CPA基因,命名为HbCPA。HbCPA序列全长1 342 bp,开放阅读框为906 bp,编码301个氨基酸。HbCPA理论分子量约为33.5 kD,理论等电点为6.01。进化分析表明,HbCPA与木薯MeCPA、蓖麻RcCPA和麻风树JcCPA聚为一枝。qRT-PCR结果显示,HbCPA在橡胶树各组织中均有表达,表达量从高到低依次为树皮、叶片、雌花、雄花、茎尖;HbCPA在叶片不同发育时期表达存在变化,淡绿期最高,古铜期和衰老期表达量相当且最低;同健康树树皮相比,HbCPA在死皮树树皮中表达量明显上调;HbCPA表达受乙烯利、茉莉酸甲酯、低温、干旱、高盐、过氧化氢等调控,表明HbCPA可能参与橡胶树产排胶调控及非生物逆境胁迫响应。

关键词: 橡胶树, N-氨甲酰基腐胺酰胺水解酶, 非生物胁迫, 生物信息学, 表达分析

Abstract:

N-carbamoylputreseine amidohydrolase（CPA）is the final reaction enzyme for higher plants to synthesize putrescine through the arginine pathway. A CPA gene from Hevea brasiliensis was cloned with RACE and RT-PCR and designated as HbCPA. The full length of HbCPA was 1 342 bp with an open reading frame of 906 bp and encoded 301 amino acids. The molecular weight and isoelectric point of HbCPA were 33.5 kD and 6.01,respectively. Phylogenetic analyses showed that HbCPA and MeCPA in Manihot esculenta,RcCPA in Ricinus communis and JcCPA in Jatropha curcas clustered into one branch. qRT-PCR analyses demonstrated that HbCPA expressed in each tissue,with the highest expression in barks,followed by leaves,female flowers,male flowers and stem apexes. The expression of HbCPA varied in different developmental stages of leaves,with the highest in the light green period,and the same and lowest expression in the bronze and senescence stages. The expression of HbCPA was significantly up-regulated in barks of tapping panel dryness（TPD）tree compared with the healthy rubber tree. HbCPA expression was also regulated by ethephon,MeJA,low temperature,drought,high salt and hydrogen peroxide. All these results suggest that HbCPA may be involved in the regulation of rubber production and debinding in rubber tree and the response to abiotic stress.

Key words: Hevea brasiliensis, N-carbamoylputreseine amidohydrolase, abiotic stress, bioinformatics analysis, expression analysis

杨洪, 胡燕玲, 岳镒繁, 邓治, 代龙军, 李德军. 橡胶树HbCPA基因的克隆、表达及生物信息学分析[J]. 生物技术通报, 2020, 36(11): 39-47.

YANG Hong, HU Yan-ling, YUE Yi-fan, DENG Zhi, DAI Long-jun, LI De-jun. Cloning,Expression and Bioinformatics Analyses of HbCPA Gene from Hevea brasiliensis[J]. Biotechnology Bulletin, 2020, 36(11): 39-47.

图/表 5

图1 HbCPA cDNA序列及其编码的氨基酸序列

图2 HbCPA与其它植物CPAs多序列比对及二级结构预测多序列比对中使用的CPA序列如下：HbCPA（Hevea brasiliensis,XP_021686375）,MeCPA（Manihot esculenta,XP_021610126）,RcAIH（Ricinus communis,XP_015577648）,JcAIH（Jatropha curcas,XP_012076119）,TcAIH（Theobroma cacao,XP_007015791）,MtCPA（Medicago truncatula,XP_003596833）,GmCPA（Glycine max,XP_003543427）,PtCPA（Populus trichocarpa,XP_006384793）,PeCPA（Populus euphratica,XP_011006805）,DzCPA（Durio zibethinus,XP_022756652）,CcCPA（Citrus clementina,XP_006424218）,CsCPA（Citrus sinensis,KDO58117）,ZjCPA（Ziziphus jujuba,XP_015872695）,AtNLP1/CPA（Arabidopsis thaliana,NP_001324215）,StCPA（Solanum tuberosum,NP_001275125）,OsCPA（Oryza sativa,EAY86155）,PaAIH（Prunus avium,XP_021810000）;序列上方标有α和η的波形曲线分别表示α-螺旋和310-螺旋;标有β的箭头表示β-单链;标有TT字母的表示α-和β-转角;二级结构预测以蒺藜苜蓿MtCPA晶体结构（PDB ID：5H8I）为模型

图3 HbCPA与其它物种CPAs蛋白系统进化分析

图4 HbCPA在橡胶树不同组织（A）、叶片不同发育时期（B）及健康/死皮橡胶树胶乳和树皮（C）表达模式

图5 不同处理条件下HbCPA的表达模式 A：H2O2;B：乙烯利;C：茉莉酸甲酯;D：低温;E：干旱;F：高盐

参考文献 28

[1]	Cornish K. Similarities and differences in rubber biochemistry among plant species[J]. Phytochemistry, 2001,57(7):1123-1134. URL pmid: 11430985
[2]	李亚栋, 何近刚. 植物多胺代谢与胁迫响应研究进展[J]. 华北农学报, 2012,27(s1):240-245. doi: 10.3969/j.issn.1000-7091.2012.z1.048 URL
	Li YD, He JG. Advance in metabolism and response to stress of polyamines in plant[J]. Acta Agriculturae Boreali-Sinica, 2012,27(s1):240-245.
[3]	沈永娟, 张辉, 王倩, 等. 植物中多胺的合成代谢及其分子生物学水平研究进展[J]. 保鲜与加工, 2018,18(6):165-169.
	Shen YJ, Zhang H, Wang Q, et al. Research progress on synjournal metabolism and molecular biology of polyamines in plants[J]. Storage and Process, 2018,18(6):165-169.
[4]	马瑛, 刘静. 植物体中多胺代谢及其功能研究进展[J]. 陕西理工学院学报:自然科学版, 2010,26(2):47-54.
	Ma Y, Liu J. Progress in metabolism and functions of polyamines in plants[J]. Journal of Shananxi University of Technology:Natural Science Edition, 2010,26(2):47-54.
[5]	Wallace HM, Fraser AV, Hughes A. A perspective of polyamine metabolism[J]. Biochemical Journal, 2003,376(1):1-14.
[6]	Kusano T, Berberich T, et al. Polyamines:essential factors for growth and survival[J]. Planta, 2008,228(3):367-381. doi: 10.1007/s00425-008-0772-7 URL pmid: 18594857
[7]	Bartosz S, Milosz R, Maura M, et al. Structural investigations of N-carbamoylputrescine amidohydrolase from Medicago truncatula:insights into the ultimate step of putrescine biosynjournal in plants[J]. Front Plant Sci, 2016,7:350. doi: 10.3389/fpls.2016.00350 URL pmid: 27066023
[8]	Hanfrey C, Sommer S, Mayer MJ, et al. Arabidopsis polyamine biosynjournal:absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity[J]. Plant J, 2001,27(6):551-560. doi: 10.1046/j.1365-313x.2001.01100.x URL pmid: 11576438
[9]	Kumar RR, Sharma SK, Rai GK, et al. Exogenous application of putrescine at pre-anjournal enhances the thermotolerance of wheat(Triticum aestivum L.)[J]. Indian J Biochem Biophys 2014,51(5):396-406. URL pmid: 25630110
[10]	Liu JH, Kazuyoshi N, Chikako H, et al. Polyamine biosynjournal of apple callus under salt stress:importance of the arginine decarboxylase pathway in stress response[J]. J Exp Bot, 2006,57(11):2589-2599. doi: 10.1093/jxb/erl018 URL pmid: 16825316
[11]	Roy M, Wu R. Arginine decarboxylase transgene expression and analysis of environmental stress tolerance in transgenic rice[J]. Plant Sci, 2001,160(5):869-875. doi: 10.1016/s0168-9452(01)00337-5 URL pmid: 11297783
[12]	Capell T, Bassie L, Christou P. Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress[J]. Proc Natl Acad Sci USA, 2004,101(26):9909-9914. doi: 10.1073/pnas.0306974101 URL pmid: 15197268
[13]	Kasukabe Y. Overexpression of spermidine synthase enhances tolerance to multiple environmental stresses and up-regulates the expression of various stress-regulated genes in transgenic Arabidopsis thaliana[J]. Plant Cell Physiol, 2004,45(6):712-722. URL pmid: 15215506
[14]	Alcázar R, Planas J, Saxena T, et al. Putrescine accumulation confers drought tolerance in transgenic Arabidopsis plants over-expressing the homologous Arginine decarboxylase 2 gene[J]. Plant Physiol Biochem, 2010,48(7):547-552. doi: 10.1016/j.plaphy.2010.02.002 URL pmid: 20206537
[15]	Wang BQ, Zhang QF, Liu JH, et al. Overexpression of PtADC confers enhanced dehydration and drought tolerance in transgenic tobacco and tomato:effect on ROS elimination[J]. Biochem Biophys Res Commun, 2011,413(1):10-16. URL pmid: 21871871
[16]	黄雪梅, 杨少琼, 等. 多胺在RRIM600橡胶树割面上的分布及与割胶的关系[J]. 热带作物学报, 2000,21(4):15-19.
	Huang XM, Yang SQ, et al. Distribution of polyamines in panel of rubber trees RRIM600 and its relationship to tapping[J]. Chinese Journal of Tropical Crops, 2000,21(4):15-19.
[17]	Zhao M, Liu H, Deng Z, et al. Molecular cloning and characterization of S-adenosylmethionine decarboxylase gene in rubber tree(Hevea brasiliensis)[J]. Physiol Mol Biol Plants, 2017,23(2):281-290. URL pmid: 28461717
[18]	侯岚菲, 杨洪, 邓治, 等. 橡胶树ADC1的克隆、表达及生物信息学分析[J]. 生物技术通报, 2018,34(11):117-125.
	Hou LF, Yang H, Deng Z, et al. Cloning, expression and bioinformatics analyses of an ADC1 gene in Hevea brasiliensis[J]. Biotechnology Bulletin, 2018,34(11):117-125.
[19]	杨洪, 岳镒繁, 胡燕玲, 等. 巴西橡胶树HbAIH基因的克隆及表达分析[J]. 生物技术通报, 2020,36(5):120-129.
	Yang H, Yue YF, Hu YL, et al. Cloning and expression analyses of HbAIH gene from Hevea brasiliensis[J]. Biotechnology Bulletin, 2020,36(5):120-129.
[20]	Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server[J]. Nucleic Acids Res, 2014,42(W1):W320-W324. doi: 10.1093/nar/gku316 URL
[21]	Chen CY, Chiu WC, Liu JS, et al. Structural basis for catalysis and substrate specificity of Agrobacterium radiobacter N-Carbamoyl-D-amino acid amidohydrolase[J]. J Biol Chem, 2003,278(28):26194-26201. doi: 10.1074/jbc.M302384200 URL pmid: 12709423
[22]	王立丰. 光照强度对巴西橡胶树淡绿期叶片光合特性和活性氧代谢的影响[J]. 热带作物学报, 2014,35(6):1131-1136.
	Wang LF. Effects of different light intensity on photosynthetic characteristics and ROS metabolism in light green leaves of rubber tree(Hevea brasiliensis Müll. Arg.)[J]. Chinese Journal of Tropical Crops, 2014,35(6):1131-1136.
[23]	庄海燕, 安锋, 张硕新, 等. 乙烯利刺激橡胶树增产机制研究进展[J]. 林业科学 2010,46(4):120-125.
	Zhuang HY, An F, Zhang SX, et al. Progress in study on the mechanisms to increase latex yield of Hevea brasiliensis by ethephon stimula[J]. Scientia Silvae Sinicae, 2010,46(4):120-125.
[24]	Hao BZ, Wu JL. Laticifer differentiation in Hevea brasiliensis:induction by exogenous jasmonic acid and linolenic acid[J]. Annals of Botany, 2000,85(1):37-43.
[25]	段翠芳, 曾日中, 黎瑜. 激素对巴西橡胶树橡胶生物合成的调控[J]. 热带农业科学, 2004,24(5):61-68.
	Duan CF, Zeng RZ, Li Y. Regulation of plant hormones on biosynjournal of natural rubber in Hevea brasiliensis[J]. Chinese Journal of Tropical Agriculture, 2004,24(5):61-68.
[26]	袁红梅, 洪灏, 黄惜. 巴西橡胶树产排胶机理的研究进展[J]. 分子植物育种, 2015,13(5):1151-1156.
	Yuan HM, Hong H, Huang X. The research advance of biosynjournal and drainage of latex in Hevea brasiliensis[J]. Molecular Plant Breeding, 2015,13(5):1151-1156.
[27]	Do PT, Drechsel O, Heyer AG, et al. Changes in free polyamine levels, expression of polyamine biosynjournal genes, and performance of rice cultivars under salt stress:a comparison with responses to drought[J]. Front Plant Sci, 2014,5(8):182.
[28]	Sen S, Ghosh D, Mohapatra S. Modulation of polyamine biosynjournal in Arabidopsis thaliana by a drought mitigating Pseudomonas putida strain[J]. Plant Physiol Biochem, 2018,129:180-188. URL pmid: 29886249