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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (12): 1882-1890.doi: 10.3724/SP.J.1006.2018.01882

• RESEARCH NOTES • Previous Articles    

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 Online:2018-12-12 Published:2018-09-18
  • Contact: Zi-Gang LIU,Qi-Xian CHEN E-mail:739015868@qq.com;cqxwin@163.com
  • 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)

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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

Fig. 1

Two-dimensional electrophoresis reference map of winter rapeseed Longyou 7 A: pH 4-7 contrast; B: pH 4-7 drought stress treatment."

Table 1

Identification of special proteins by LC-MS"

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

Fig. 2

Nucleotide sequence and deduced amino acid sequence of rbcL and rbcS cDNA An upstream start codon ATG is boxed; * indicates the positions of termination codon."

Fig. 3

rbcL and rbcS protein tertiary structure and active pocket in winter rapeseed A : rbcL protein tertiary structure; B: rbcS protein tertiary structure; purple shows differential pockets."

Fig. 4

Phylogenetic tree of rbcL and rbcS proteins with other related species"

Fig. 5

Semi-quantitative RT-PCR analysis and relative expression of rbcL and rbcS genes in winter rapeseed under drought stress CK: control; MR: middle drought stress."

Table 2

Photosynthetic gas exchange parameters and RuBPcase activity of winter rapeseed ‘Longyou 7’ under drought stress"

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
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