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作物学报 ›› 2020, Vol. 46 ›› Issue (10): 1507-1516.doi: 10.3724/SP.J.1006.2020.04015

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

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

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   

  1. 甘肃省干旱生境作物生物学重点实验室 / 甘肃省作物遗传改良与种质创新重点实验室 / 甘肃省油菜工程与技术研究中心 / 甘肃农业大学农学院, 甘肃兰州 730070
  • 收稿日期:2020-01-17 接受日期:2020-04-15 出版日期:2020-10-12 网络出版日期:2020-05-14
  • 通讯作者: 刘自刚
  • 作者简介:E-mail: 1291402843@qq.com
  • 基金资助:
    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(2018YFD0100502-2);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(17ZD2NA016-4)

Differential proteomics analysis of fertility transformation of the winter rape thermo-sensitive sterile line PK3-12S (Brassica rapa L.)

MI Wen-Bo(), FANG Yuan, LIU Zi-Gang*(), XU Chun-Mei, LIU Gao-Yang, ZOU Ya, XU Ming-Xia, ZHENG Guo-Qiang, CAO Xiao-Dong, FANG Xin-Ling   

  1. Gansu Provincial Key Laboratory of Arid Land Crop Sciences / Key Laboratory of Crop Genetics Improvement and Germplasm Enhancement of Gansu Province / Gansu Research Center of Rapeseed Engineering and Technology / College of Agronomy, Gansu Agricultural University, Lanzhou 730070, Gansu, China
  • Received:2020-01-17 Accepted:2020-04-15 Published:2020-10-12 Published online:2020-05-14
  • Contact: Zi-Gang LIU
  • Supported by:
    National Natural Science Foundation of China(31660404);National Key Basic Research Development Program(2018YFD0100502-2);Gansu University Scientific Research Achievement Transformation and Cultivation Project(2018D-13);National Modern Agricultural Industry Technology System Construction Project(CARS-13);Gansu Science and Technology Major Special Project(17ZD2NA016-4)

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摘要：

为揭示温敏不育系PK3-12S育性转换机制, 本研究以白菜型冬油菜温敏不育系PK3-12花药为材料, 采用2-DE和LC-MS/MS质谱鉴定等差异蛋白组学方法, 分离鉴定了PK3-12在不育/可育条件下花药差异表达蛋白质, 并对差异表达蛋白进行了生物信息学分析; 进而采用RT-PCR检测了PK3-12在不育/可育条件下花蕾发育进程中差异蛋白编码基因表达量变化。结果表明, 高温不育条件下, PK3-12花药形态瘪小, 药室有少量败育花粉, 育性转换受1对隐性基因控制, 表达变化量在2倍以上差异蛋白质点31个, 其中增量表达蛋白质点6个, 减量表达蛋白质点11个, 表达完全抑制蛋白点12个, 不育花药特异表达蛋白点2个。质谱鉴定出15个差异蛋白质, 参与信号转导通路、二羧基乙醛酸代谢、糖酵解代谢、次生合成代谢、氨基酸生物合成、分支酸生物合成、碳代谢途径等细胞过程。Rubisco亚基连接蛋白编码基因BrrbcL开放读码框(open reading frame, ORF)长度为1095 bp, 编码364个氨基酸; 与可育花蕾相比, 发育进程中不育花蕾BrrbcL基因、膜联蛋白基因(ANN)、BetVI过敏原家族基因(BetVI)表达明显下调, 表明上述基因可能参与了温敏不育系PK3-12S育性的转换。

关键词: 白菜型冬油菜, 温敏不育, 差异蛋白组学, 基因表达

Abstract:

To reveal the fertility switching mechanism of temperature-sensitive sterility line PK3-12S (Brassica rapa L.), the differentially expressed proteins were isolated and identified using anthers of PK3-12S in sterile/fertile conditions by 2-DE and LC-MS/MS mass spectrometry. The expression level variations of differentially expressed genes were examined by RT-PCR in PK3-12 flower buds during sterility/fertile development. The result showed that the sterile anther size of PK3-12 was small with a little abortive pollen in the anther room under high temperature. The trait of fertility transformation was controlled by a pair of recessive alleles. There were 31 differentially expressed proteins with more than two times of the expression level, including six protein spots with increasing expression, 11 protein spots with reduced expression, 12 protein spots with complete inhibition, and two protein spots with induced expressed. Fifteen differentially expressed proteins involved in the cellular processes such as signal transduction pathways, glyoxylate and dicarboxylate metabolism, glycolysis gluconeogenesis, biosynthesis of secondary metabolites, biontheses of amino acids, chorismate biosynthesis, and carbon metabolism pathways were identified by mass spectrometry. The BrrbcL gene, encoding a Rubisco subunit-binding accessory protein, had an open reading frame (ORF) in length of 1095 bp encoded 364 amino acids. Compared with fertile anthers, the expression level of BrrbcL gene, annexin gene (ANN) and BetVI allergen family gene (BetVI) was significantly down-regulated during sterile anthers development, which indicated that these genes maybe participate in the fertility transformation of the thermo-sensitive sterile line PK3-12S.

Key words: winter turnip rape (Brassica rapa L.), thermo-sensitive sterile line, proteomics, gene expression

表1

差异表达基因定量引物"

基因
Gene		 引物序列
Primer sequence (5°-3°)		 产物长度
Product length (bp)
Rbcl F: CAGTCCCAGCTACGACCTTCT 134
R: CCTGTCTCCATCGGTTTGTTT
Ann F: CCGGAACAGACGAAGGAGCT 169
R: TCACCGAGAAGTGCGACGAG
CTIMC F: CAGCCCAAGCTCAGGAAGTA 166
R: CCACCGACCAAGAAACCATC
Bet VI F: CCCCACTGGTGAAAGTATCGG 138
R: CCTTGGGAGTAACGGTGATGG

图1

白菜型冬油菜热敏感不育系PK3-12S可育(A)和不育(B)花形态的比较"

表2

PK3-12S组合后代育性表现"

组合
Combination		 可育株数/株
Number of fertile plants/ plant		 不育株数/株
Number of sterile plants/ plant		 分离比
Separation ratio		 卡方测验
Chi-square test
F1 F2 BC1 F1 F2 BC1 F2 BC1 F2 BC1
PK3-12S×QX6-3 26
31
42		 65
86
47		 34
63
39		 0
0
0		 21
31
14		 36
56
32		 3.10:1 0.94:1 3:1 1:1
PK3-12S×QX21-2 2.77:1 1.13:1 3:1 1:1
PK3-12S×LX2-3 3.36:1 1.22:1 3:1 1:1

图2

白菜型冬油菜生态不育系PK3-12可育时期(左)及其不育时期(右)花器全蛋白双向电泳图谱"

表3

PK3-12S可育时期与不育时期花器差异表达蛋白点质谱鉴定结果"

编号
No.		 登录号
Accession No.		 蛋白名称
Protein name		 调节
Regulated		 物种来源
Plant species		 蛋白分子量
Protein MW		 多肽片
段数
Pep. count		 蛋白得分
Protein score CI (%)
1 A0A078JSU1 3-磷酸草莽酸-1-乙烯基乙酰羧化转移酶
3-phosphoshikimate-1-carboxyvinyltransferase		 + Brassica napus 55720.4 18 100
2 M1F2H2 1,5-二磷酸核酮糖羧化/加氧酶
Chloroplast ribulose-1,5-bisphosphate		 - Brassica oleracea 47858.1 10 99.999
3 A0A0D3E3Z1 琥珀酰辅酶A连接酶β亚基
Succinyl-CoA ligase subunit beta		 - Brassica oleracea 45214.0 17 100
4 M4CHC2 磷酸甘油酸激酶
Phosphoglycerate kinase		 - Brassica rapa subsp. 42368.6 16 100
9 M4F009 膜联蛋白
Annexin		 - Brassica rapa subsp. pekinensis 37045.8 15 99.618
11 A0A078GNP3 硫氧还蛋白还原酶
Thioredoxin reductase		 - Brassica napus 72670.1 12 99.821
12 M4CN89 铁蛋白
Ferritin		 - Brassica rapa subsp. pekinensis 30584.2 10 100
16 U5IBV8 谷胱甘肽巯基转移酶
Glutathion-S-transferase taub (fragment)		 + Brassica oleracea 17916.5 9 100
17 M4DE25 磷酸丙糖异构酶
Triosephosphate isomerase		 - Brassica rapa subsp. 27221.1 13 100
18 E5KXU6 超氧化物歧化酶
Superoxide dismutase (fragment)		 - Brassica campestris 22151.1 3 100
19 Q944W6 肿瘤翻译调控因子同源蛋白
Translationally-controlled tumor protein homolog		 - Brassica oleracea 19027.6 8 96.594
20 O82795 热应答蛋白
Heat stress-induced protein		 + Brassica oleracea 23474.3 6 100
26 A8IXG5 BetVI过敏原家族蛋白
BetVI allergen family protein		 + Brassica campestris 17149.9 12 100
27 M4E5U4 抑制蛋白
Profilin		 - Brassica rapa subsp. pekinensis 14518.1 5 100
30 A1YN07 Kunitz型半胱氨酸蛋白酶抑制因子
Kunitz-type cysteine protease inhibitor		 + Brassica campestris 24744.6 6 100

图3

差异蛋白GO功能分析"

图4

差异表达蛋白KEGG分类图"

图5

PK3-12S花器发育过程中差异蛋白编码基因的相对表达量 rbcL: 1,5-二磷酸核酮糖羧化酶; ANN: 膜联蛋白; CTIMC: 磷酸丙糖异构酶; BetVI: BetVI过敏原家族蛋白。"

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