摘要
应用双向电泳结合液相色谱-质谱联用(LC-MS)分析,筛选得到干旱胁迫下白菜型冬油菜与光合作用相关差异蛋白质RuBisCo小亚基。根据已发表白菜型油菜(Brassicarapa)RuBisCo亚基rbcL和rbcS保守序列设计引物,采用RT-PCR扩增陇油7号的cDNA,获得rbcL和rbcS基因开放阅读框,长度分别为1095 bp和549 bp,编码364和181个氨基酸的蛋白质。生物信息学分析结果显示, rbcL与大白菜(Brassica rapa subsp. chinensis)的蛋白质同源性达到99%以上,其保守序列属于RuBisCoLarge超家族,该蛋白质理论相对分子量及等电点分别为40.29kDa和6.70,不稳定指数(instabilityindex,II>40为不稳定蛋白质)为41.67,属于不稳定蛋白;脂融指数(aliphaticindex)为83.63,平均亲水性值(grand average of hydropathicity)为-0.232,属于亲水性蛋白质;二级结构包括38.74%α-螺旋(alpha helix)、10.99%延伸链(extendedstrand)及50.27%自由卷曲(randomcoil);三级结构具有5个不同的活性口袋。rbcS与甘蓝(Brassicaoleracea)蛋白质同源性达到99%,其保守序列具有rbcS超家族和RuBisCo Small Like超家族,理论相对分子量为20.32 kDa,理论等电点为8.23,不稳定指数为33.66,属于稳定蛋白质;脂融指数为74.86,平均亲水性值为-0.142,属于亲水性蛋白质;二级结构包括16.02%α-螺旋、28.73%的延伸链及55.25%自由卷曲;三级结构具有4个不同的活性口袋。实时荧光定量和半定量分析显示,干旱胁迫下,白菜型冬油菜叶片rbcL和rbcS基因表达量下调是光合作用下降的原因。并且,冬油菜叶片Pn下降与RuBPCase表达抑制和活性降低有关,非气孔限制是Pn下降的主要因素。
In this study, we used the two-dimensional gel electrophoresis(2 D-DIGE) technology combined with the liquid chro-matography-mass spectrometry(LC-MS) technology to filtrate the differential protein small subunit of RuBisCo which was related to photosynthesis in winter rapeseed(Brassica rapa) under drought stress. According to the published Brassica rapa RuBisCo subunit conserved sequences of rbcL and rbcS, we designed the primers and used the reverse transcription-polymerase chain reaction(RT-PCR) technology to amplify the cDNA sequence in Longyou 7. We obtained the open reading frame(ORF) of the RuBisCo subunit rbcL and rbcS, which had the length 1095 bp and 549 bp, and encoded the proteins contains 364 and 181 amino acids, respectively. The results of bioinformatics analysis showed that compare with Brassica rapa subsp. chinensis and Brassica rapa, the protein homology of rbcL and in Longyou 7 was above 99%, with the conserved domain sequence belonging to RuBisCo large superfamily. The theoretical relative molecular mass and isoelectric point of rbcL were 40.29 kDa and 6.70, respectively. And the instability index was-41.67(II> 40 was considered as unstable protein), showing that it was an unstable protein. The aliphatic index was 83.63 and the grand average of hydropathicity was 0.232, which indicated that it was a hydrophilic protein. The secondary structure included 38.74% alpha helix, 10.99% extended strand and 50.27% random coil. And the tertiary structure contained five differential activity pockets. The protein homology of rbcS and Brassica oleracea in Longyou 7 was 99%, that conserved domain sequence was contained the rbcS superfamily and the RuBisCo Small like superfamily, which theoretical relative molecular mass and isoelectric point were 20.32 kDa and 8.23, respectively. The instability index was 33.66, showing that was a stable protein, and the aliphatic index was 74.86, the grand average of hydropathicity was-0.142, showing a hydrophilic protein. The secondary structure included 16.02% alpha helix, 28.37% extended strand and 55.25% random coil. And the tertiary structure contained four differential activity pockets. Real-time quantitative and semi-quantitative results showed that, the expression of rbcL and rbcS in winter rapeseed leaves under drought stress was down-regulated, which was the reason of decreasing photosynthesis. In addition, the decreased net photosynthesis rate(Pn) in winter rapeseed leaves was related to the inhibited RuBPCase expression and decreased RuBPCase activity, and the non-stomatal limitation was the main factor of declined Pn.
引文
[1]张兴华,高杰,杜伟莉,张仁和,薛吉全.干旱胁迫对玉米品种苗期叶片光合特性的影响.作物学报,2015,41:154-159Zhang X H,Gao J,Du W L,Zhang R H,Xue J Q.Effects of drought stress on photosynthetic characteristics of maize hybrids at seedling stage.Acta Agron Sin,2015,41:154-159(in Chinese with English abstract)
[2]Jeroni G,Miquel R C,Hipólito M,Jaume F.Rubisco activity in Mediterranean species is regulated by the chloroplastic CO2concentration under water stress.J Exp Bot,2011,62:653-665
[3]Massacci A,Nabiev S M,Pietrosanti L,Nematov S K,Chernikova T N,Thor K,Leipner J.Response of the photosynthetic apparatus of cotton(Gossypium hirsutum)to the onset of drought stress under field conditions studied by gas-exchange analysis and chlorophyll fluorescence imaging.Plant Physiol Biochem,2008,46:189-195
[4]Flexas J,Ribas-CarbóM,Bota J,Galmés J,Henkle M,Martínez-Ca?ellas S,Medrano H.Decreased Rubisco activity during water stress is not induced by decreased relative water content but related to conditions of low stomatal conductance and chloroplast CO2 concentration.New Phytol,2006,172:73-82
[5]Ma P D,Lu T C,Zhou X F,Zhu X J,Wang X Z.Preparation of polyclonal antibodies of Rubisco large and small subunits and their application in the functional analysis of the genes.Acta Biochim Biophys Sin,2004,36:644-648
[6]王玉华,吴忠义,贾敬芬,张秀海,黄丛林.甘薯叶绿体rbcL基因的克隆与序列分析.植物生理学通讯,2008,44:882-886Wang Y H,Wu Z Y,Jia J F,Zhang X H,Huang C L.Cloning and sequence analyses of the rbcL gene from chloroplast of sweet potato[Ipomoea batatas(L.)Lam.].Plant Physiol J,2008,44:882-886(in Chinese with English abstract)
[7]刘晓庆,崔喜艳,丁志鑫,李海霞.大豆rbcL基因克隆、序列分析及原核表达.中国油料作物学报,2011,33:226-230Liu X Q,Cui X Y,Ding Z X,Li H X.Cloning,sequence analysis and prokaryotic expression of rbc L gene from Glycine max.Chin JOil Crop Sci,2011,33:226-230(in Chinese with English abstract)
[8]齐国昌.青稞耐盐性品种(系)的筛选及rbcS基因的克隆与表达分析.四川农业大学硕士学位论文,四川成都,2014Qi G C.Salt Tolerance Qingke Varieties(lines)Screening and rbcS Gene Cloning and Its Expression Snalysis in Qingke(Hulless Barley).MS Thesis of Sichuan Agricultural University,Chengdu,China,2014(in Chinese with English abstract)
[9]熊勇,赵春艳,杨青松,张维汉.雏菊rbcL基因的克隆及其生物信息学分析.北方园艺,2015,(4):89-95Xiong Y,Zhao C Y,Yang Q S,Zhang W H.Molecular cloning,bioinformatics snalysis of rbcL gene in Bellis perennis.Nor Hortic,2015,(4):89-95(in Chinese with English abstract)
[10]丁帅,熊勇,李正涛,王忠诚,孟亚媛,李乐,肖焱波.菊花rbcL基因电子克隆及生物信息学、适应性进化分析.种子,2015,34(10):24-30Ding S,Xiong Y,Li Z T,Wang Z C,Meng Y Y,Li L,Xiao Y B.In silico cloning,bioinformatics and adaptive evolution analysis of rbcL gene in Chrysanthemum morifolium.Seed,2015,34(10):24-30(in Chinese with English abstract)
[11]崔大练,马玉心,石戈,范美华,杜巍,张明.紫穗槐幼苗叶片对不同干旱梯度胁迫的生理生态响应.水土保持研究,2010,17(2):178-181Cui D L,Ma Y X,Shi G,Fan M H,Du W,Zhang M.Ecophysiological responses of Amorpha fruticosa L.seeding leaves to long-term drought gradient treatment.Res Soil Water Conserv,2010,17(2):178-181(in Chinese with English abstract)
[12]梁建萍,贾小云,刘亚令,吴云,周然,冯前进.干旱胁迫对蒙古黄芪生长及根部次生代谢物含量的影响.生态学报,2016,36:4415-4422Liang J P,Jia X Y,Liu Y L,Wu Y,Zhou R,Feng Q J.Effects of drought stress on seeding growth and accumulation of secondary metabolites in the roots of Astragalus membranaceus var.mongholicus.Acta Ecol Sin,2016,36:4415-4422(in Chinese with English abstract)
[13]龚富生,张嘉宝.植物生理学实验(第2版).北京:高等教育出版社,1995.pp 90-92Gong F S,Zhang J B.Plant Physiology Experiment,2nd edn.Beijing:Higher Education Press,1995.pp 90-92(in Chinese)
[14]Bradford M M.A rapid method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem,1976,72:248-254
[15]Katayama H,Nagasu T,Oda Y.Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.Rapid Commun Mass Spectrom,2001,15:1416-1421
[16]Lawlor D W,Cornic G.Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants.Plant Cell Environ,2002,25:275-294
[17]Tezara W,Mitchell V J,Driscoll S D,Lawlor D W.Water stress inhibits plant photosynthesis by decreasing coupling factor and ATP.Nature,1999,401:914-917
[18]Yue H Y,Yin J R,Yan S Q,Feng Y L,Zhang L J,Guo J Q,Li HL,Ding X M,Shen J L.Cloning and sequence analysis of rbcSgene of wild barley(Hordeum brevisubulatum)under salt stress.Agric Sci Technol,2010,11:42-44
[19]朱磊,杨景华,张明方.芥菜Rubisco小亚基的基因克隆及其在芜菁花叶病毒侵染后的表达分析.核农学报,2011,25:945-950Zhu L,Yang J H,Zhang M F.Cloning of Rubisco small subunit gene from mustard and analysis of its expression in response to Turnip mosaic virus infection.J Nucl Agric Sci,2011,25:945-950(in Chinese with English abstract)
[20]冯玖玲.杜氏盐藻RbcS基因家族新成员的克隆及功能研究.郑州大学硕士学位论文,河南郑州,2010Feng J L.Cloning and Functional Analysis of a New Member of RbcS Gene Family from Dunaliella salina.MS Thesis of Zhengzhou University,Zhengzhou,China,2010(in Chinese with English abstract)
[21]Mccourt R M,Karol K G,Kaplan S,Hoshaw R W.Using rbcLsequences to test hypotheses of chloroplast and thallus evolution in conjugating green algae(Zygnematales,charophyceae).JPhycol,2010,31:989-995
[22]Nozaki H,Ito M,Uchida H,Watanabe M M,Kuroiwa T.Phylogenetic analysis of eudorina species(Volvocaceae,chlorophyta)based on rbcL gene sequences.J Phycol,2010,33:859-863
[23]朱海林,刘秀明,江莺,杜美丽,李巍,李海燕,李校堃.苜蓿叶绿体DNA的提取及其RbcL基因片段的克隆.安徽农业科学,2011,39:13306-13308Zhu H L,Liu X M,Jiang Y,Du M L,Li W,Li H Y,Li X K.Extraction of alfalfa chloroplast DNA and cloning of RbcL gene fragment.J Anhui Agric Sci,2011,39:13306-13308(in Chinese with English abstract)
[24]Bouma W L M,Ritchie P,Perrie L R.Phylogeny and generic taxonomy of the New Zealand Pteridaceae ferns from chloroplast rbcL DNA sequences.Aust Syst Bot,2010,23:143-151