欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2018, Vol. 44 ›› Issue (12): 1882-1890.doi: 10.3724/SP.J.1006.2018.01882

• 研究简报 • 上一篇    

白菜型冬油菜RuBisCo蛋白亚基基因rbcL和rbcS的克隆及其在干旱胁迫下的表达

米超2,赵艳宁2,刘自刚1,2,*(),陈其鲜3,*(),孙万仓1,2,方彦1,李学才1,2,武军艳1,2   

  1. 1甘肃省油菜工程技术研究中心 / 甘肃省干旱生境作物学重点实验室 / 甘肃省作物遗传改良与种质创新重点实验室, 甘肃兰州 730070
    2甘肃农业大学农学院, 甘肃兰州730070
    3甘肃省农业技术推广总站, 甘肃兰州730040
  • 收稿日期:2017-09-21 接受日期:2018-08-20 出版日期:2018-12-12 网络出版日期:2018-09-18
  • 通讯作者: 刘自刚,陈其鲜
  • 基金资助:
    本研究由国家自然科学基金项目(31660404);国家重点基础研究发展计划项目(2018YFD0100502);甘肃省高校科研成果转化培育项目(2018D-13);国家现代农业产业技术体系建设专项(CARS-13);甘肃省科技重大专项项目资助(17ZD2NA016-4)

Cloning of RuBisCo Subunits Genes rbcL and rbcS from Winter Rapeseed (Brassica rapa) and Their Expression under Drought Stress

Chao MI2,Yan-Ning ZHAO2,Zi-Gang LIU1,2,*(),Qi-Xian CHEN3,*(),Wan-Cang SUN1,2,Yan FANG1,Xue-Cai LI1,2,Jun-Yan WU1,2   

  1. 1 Gansu Research Center of Rapeseed Engineering and Technology / Improvement and Key Laboratory of Crop Genetics and Germplasm Enhancement of Gansu Province / Gansu Provincial Key Laboratory of Arid Land Crop Sciences, Lanzhou 730070, Gansu, China
    2 College of Agronomy, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    3 Gansu General Station of Agro-technology Extension, Lanzhou 730070, Gansu, China
  • Received:2017-09-21 Accepted:2018-08-20 Published:2018-12-12 Published online:2018-09-18
  • Contact: Zi-Gang LIU,Qi-Xian CHEN
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31660404);the National Key Research and Development Program of China(2018YFD0100502);Transformation and Cultivation of Scientific Research Achievements in Universities in Gansu(2018D-13);Special Funds for the Construction of Modern Agricultural Technology System in Gansu, the National Modern Agro-industry Technology System(CARS-13);Gansu Science and Technology Major Project(17ZD2NA016-4)

RichHTML

6

PDF

855

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(Brassica rapa) 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RuBisCo Large超家族, 该蛋白质理论相对分子量及等电点分别为40.29 kDa和6.70, 不稳定指数(instability index, II>40为不稳定蛋白质)为41.67, 属于不稳定蛋白; 脂融指数(aliphatic index)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(extended strand)及50.27%自由卷曲(random coil); 三级结构具有5个不同的活性口袋。rbcS与甘蓝(Brassica oleracea)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。

关键词: 白菜型冬油菜, rbcL, rbcS, 干旱胁迫

Abstract:

In this study, we used the two-dimensional gel electrophoresis (2D-DIGE) technology combined with the liquid chromatography-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.

Key words: winter rapeseed (Brassica rapa), rbcL, rbcS, drought stress

图1

白菜型冬油菜陇油7号双向电泳图 A: pH 4~7对照; B: pH 4~7干旱处理。"

表1

LC-MS鉴定结果"

序号
Spot No.		 登录号
Accession		 蛋白名称
Protein name		 分子量/等电点
MW/pI		 得分
Score		 来源
Resource		 上调/下调
Up/down
1 gi|541620 低温相关蛋白BN115
Low-temperature regulated protein BN115		 14.8 kDa/7.75 81.8 甘蓝型油菜
Brassica napus		 +10
2 gi|229893642 病毒抗性蛋白
Virus-resistance protein		 20.1 kDa/7.31 46.1 大白菜
Brassica rapa subsp. chinensis		 +20
3 gi|109389998 叶绿体叶绿素a/b结合蛋白
Chloroplast chlorophyll a/b binding protein		 28.3 kDa/5.22 46.2 甘蓝型油菜
Brassica napus		 +12
4 gi|132091 叶绿体Rubisco F1小链
Ribulose bisphosphate carboxylase small
chain F1, chloroplastic; Short		 20.2 kDa/7.31 111 甘蓝型油菜
Brassica napus		 -10
5 gi|171702843 半胱氨酸蛋白酶
Cysteine protease		 50.5 kDa/5.17 73.7 白菜型油菜
Brassica rapa		 +18
6 gi|19570344 核苷二磷酸激酶1
Nucleoside diphosphate kinase 1		 16.4 kDa/6.05 101 白菜型油菜
Brassica rapa		 -11
7 gi|15225120 应激蛋白α-β结构域
Stress responsive alpha-beta barrel domain
protein		 22.5 kDa/5.04 135 拟南芥
Arabidopsis thaliana		 +13
8 gi|15241956 谷胱甘肽S-转移酶家族蛋白
Glutathione S-transferase family protein		 31.9 kDa/8.03 79.1 拟南芥
Arabidopsis thaliana		 -15
序号
Spot No.		 登录号
Accession		 蛋白名称
Protein name		 分子量/等电点
MW/pI		 得分
Score		 来源
Resource		 上调/下调
Up/down
9 gi|356494238 磷酸甘露糖异构酶
Phosphomannomutase		 27.9 kDa/5.23 95.6 大白菜
Brassica rapa subsp. chinensis		 +13
10 gi|29778743| 叶绿体β-碳酸酐酶
Chloroplast beta-carbonic anhydrase		 37.7kDa/6.00 106 甘蓝型油菜
Brassica napus		 +11
11 gi|14009294 假定的6-磷酸葡萄糖酸内酯酶
Putative 6-phosphogluconolactonase		 29.3 kDa/5.90 114 埃塞俄比亚芥
Brassica carinata		 +14
12 gi|15234962 DNAJ热激蛋白N-端结构域
DNAJ heat shock N-terminal
domain-containing protein		 38.4 kDa/7.38 50.8 拟南芥
Arabidopsis thaliana		 +12
13 gi|211905345 上皮硫特异蛋白
Epithiospecifier protein		 37.9kDa/5.71 73.8 白菜
Brassica rapa subsp. pekinensis		 +15
14 gi|62321480 脂氧合酶
Lipoxygenase		 103.3 kDa/5.36 96.4 拟南芥
Arabidopsis thaliana		 -14
15 gi|89257686 假定的乙酰鸟苷酸脱乙酰基酶
Acetylornithine deacetylase, putative		 48.0 kDa/5.62 129.0 甘蓝
Brassica oleracea		 +12
16 gi|19553697| β-1,3-葡聚糖酶
Beta-1,3-glucanase		 38.9 kDa/4.89 95.6 大白菜
Brassica rapa subsp. chinensis		 +16
17 gi|356494248 L-半乳糖脱氢酶
L-galactose dehydrogenase		 34.9 kDa/5.08 68 大白菜
Brassica rapa subsp. chinensis		 -9
18 gi|41584275 热休克同源蛋白70
Heat shock cognate protein 70		 28.1 kDa/5.17 65.7 山嵛菜
Eutrema halophilum		 -16
19 gi|28540855 可溶性淀粉合成酶
Soluble starch synthase		 71.6 kDa/5.24 103 白菜
Brassica rapa subsp. pekinensis		 +17
20 gi|15229519 二羟丙酮激酶
Dihydroxyacetone kinase		 61.9 kDa/5.22 111 拟南芥
Arabidopsis thaliana		 -11
21 gi|197245081 玉米黄质环氧酶
Zeaxanthin epoxidase		 79.8 kDa/5.67 108 甘蓝型油菜
Brassica napus		 -13

图2

rbcL和rbcS基因的编码区核酸序列及编码氨基酸序列 上游的起始密码子ATG用方框表示; *为终止密码的位置。"

图3

白菜型冬油菜rbcL和rbcS蛋白三级结构及活性口袋 A: rbcL蛋白质三级结构; B: rbcS蛋白质三级结构; 紫色箭头表示不同活性口袋。"

图4

rbcL和rbcS蛋白与其他相关物种蛋白序列的系统进化树"

图5

干旱胁迫下白菜型冬油菜rbcL和rbcS基因的半定量RT-PCR分析及相对表达量 CK: 对照组; MR: 中度干旱组。"

表2

干旱胁迫对白菜型冬油菜‘陇油7号’光合气体交换参数及RuBPcase活性的影响"

处理
Treatment		 气孔导度
Gs (mol H2O m-2 s-1)		 胞间CO2浓度
Ci (μmol mol-1)		 蒸腾速率
Tr (mmol H2O m-2 s-1)		 净光合速率
Pn (μmol CO2 m-2 s-1)		 RuBPCase activity
(μmol m-2 s-1)
CK 0.29±0.01 a 330.4±6.65 b 6.63±0.18 a 0.90±0.04 a 35.05±0.91 a
MR 0.15±0.01 b 395.7±3.33 a 2.96±0.23 b 0.47±0.11 b 18.43±0.56 b
[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, 2015,41:154-159
doi: 10.3724/SP.J.1006.2015.00154
Zhang 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)
doi: 10.3724/SP.J.1006.2015.00154
[2] Jeroni G, Miquel R C, Hipólito M, Jaume F . Rubisco activity in Mediterranean species is regulated by the chloroplastic CO2 concentration 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
doi: 10.1016/j.plaphy.2007.10.006 pmid: 18053735
[4] Flexas J, Ribas-Carbó M, Bota J, Galmés J, Henkle M, Martínez-Cañellas 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
doi: 10.1111/nph.2006.172.issue-1
[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
doi: 10.1093/abbs/36.9.644
[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, 2008,44:882-886
Wang 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, 2011,33:226-230
Liu X Q, Cui X Y, Ding Z X, Li H X . Cloning, sequence analysis and prokaryotic expression of rbcL gene from Glycine max. Chin J Oil 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, 2014
Qi 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):89-95
Xiong 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):24-30
Ding 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):178-181
Cui 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, 2016,36:4415-4422
doi: 10.5846/stxb201412162507
Liang 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)
doi: 10.5846/stxb201412162507
[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 90-92
Gong 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
doi: 10.1016/0003-2697(76)90527-3
[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
doi: 10.1002/(ISSN)1097-0231
[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
doi: 10.1046/j.0016-8025.2001.00814.x pmid: 11841670
[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
doi: 10.1038/44842
[18] Yue H Y, Yin J R, Yan S Q, Feng Y L, Zhang L J, Guo J Q, Li H L, Ding X M, Shen J L . Cloning and sequence analysis of rbcS gene 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, 2011,25:945-950
Zhu 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, 2010
doi: 10.7666/d.y1832982
Feng 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)
doi: 10.7666/d.y1832982
[21] Mccourt R M, Karol K G, Kaplan S, Hoshaw R W . UsingrbcL sequences to test hypotheses of chloroplast and thallus evolution in conjugating green algae(Zygnematales, charophyceae). J Phycol, 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, 2011,39:13306-13308
doi: 10.3969/j.issn.0517-6611.2011.22.020
Zhu 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 ofRbcL gene fragment. J Anhui Agric Sci, 2011,39:13306-13308 (in Chinese with English abstract)
doi: 10.3969/j.issn.0517-6611.2011.22.020
[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
[1] 米文博, 方园, 刘自刚, 徐春梅, 刘高阳, 邹娅, 徐明霞, 郑国强, 曹小东, 方新玲. 白菜型冬油菜温敏不育系PK3-12S育性转换的差异蛋白质组学分析[J]. 作物学报, 2020, 46(10): 1507-1516.
[2] 马骊,袁金海,孙万仓,刘自刚,曾秀存,武军艳,方彦,李学才,陈奇,许耀照,蒲媛媛,刘海卿,杨刚,刘林波. 白菜型冬油菜类甜蛋白的筛选、克隆及其在低温胁迫下的表达[J]. 作物学报, 2017, 43(04): 620-628.
[3] 许耀照,曾秀存,张芬琴,孙佳,孙万仓,武军艳,方彦,刘自刚,孙柏林. 白菜型冬油菜叶片结构和光合特性对冬前低温的响应[J]. 作物学报, 2017, 43(03): 432-441.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2