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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (10): 1507-1516.doi: 10.3724/SP.J.1006.2020.04015

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2020-10-12 Published:2020-05-14
  • Contact: Zi-Gang LIU E-mail:1291402843@qq.com;lzgworking@163.com
  • 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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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

Table 1

Primers of some differentially expressed genes"

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

Fig. 1

Comparison of fertile (A) and sterile (B) flower morphology of thermo-sensitive sterile line PK3-12S in winter turnip rape"

Table 2

Fertility performance of PK3-12S combinations"

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

Fig. 2

Two-dimensional protein electrophoresis profiles between period (left) and sterile period (right) of ecological male sterile line PK3-12 fertile flower in Winter Turnip Rape"

Table 3

Summary of differentially expressed protein of fertile period and sterile period in PK3-12"

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

Fig. 3

GO function analysis of differentially expression proteins"

Fig. 4

KEGG pathway analysis of differentially expressed proteins"

Fig. 5

Relative expression levels of the genes encoded differentially expressed proteins during flower development of PK3-12S rbcL: ribulose-1,5-bisphosphate carboxylase; ANN: annexin; CTIMC: triosephosphate isomerase; BetVI: BetVI allergen family protein."

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