甘蓝型油菜<i>P5CR</i>同源基因的克隆表达及其多态性分析

马秀奇; 张晓娟; 孙晓敏; 陈乔; 张羽; 何开罡; 权莹; 宋建民

doi:10.19303/j.issn.1008-0384.2022.06.006

甘蓝型油菜P5CR同源基因的克隆表达及其多态性分析

doi: 10.19303/j.issn.1008-0384.2022.06.006

1.
陕西理工大学生物科学与工程学院，陕西　汉中　723001
2.
汉中市农业技术推广与培训中心，陕西　汉中　723001
3.
汉中职业技术学院，陕西　汉中　723001
4.
陕西省资源生物重点实验室，陕西　汉中　723000

基金项目: 陕西省科技计划项目（2020NY-068）；陕西理工大学人才启动项目（SLGRCQD2115）；陕西理工大学 2020年大学生创新创业训练计划项目（S202010720022）；陕西省教育厅科研项目（11JS034）

详细信息

作者简介:
马秀奇（2000−），男，硕士研究生，主要从事油菜分子遗传研究（E-mail：mxq20000308@163.com）

通讯作者:
张晓娟（1982−），女，副教授，博士，主要从事油菜分子遗传研究（E-mail：zxj12162001@163.com）

中图分类号: S 562
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出版历程
- 收稿日期: 2022-03-08
- 修回日期: 2022-04-27
- 网络出版日期: 2022-06-20
- 刊出日期: 2022-06-28

Cloning, Expression, and Polymorphism of Homologous Brassica napus P5CR

1.
School of Biological Sciences and Engineering, Shaanxi University of Technology, Hanzhong, Shaanxi　723001, China
2.
Hanzhong Agricultural Technology Extension and Training Center, Hanzhong, Shaanxi　723001, China
3.
Hanzhong Vocational and Technical College, Hanzhong, Shaanxi　723001, China
4.
Shaanxi Provincial Key Laboratory of Biological Resources, Hanzhong, Shaanxi　723000, China

摘要

摘要: 目的克隆油菜菌核病抗性相关基因，进一步为油菜抗病分子标记开发以及通过分子标记辅助育种途径选育抗菌核病油菜新品种提供理论基础。方法以高抗、高感菌核病油菜为研究材料，对甘蓝型油菜A03、C03染色体上的P5CR（Pyrroline-5-carboxylate reductase，吡咯林-5-羧酸还原酶）同源基因进行特异PCR扩增，克隆和测序及表达分析。利用DNAMAN软件对测序结果进行序列比对，寻找抗、感材料中的差异SNP位点，并分析这些位点与油菜菌核病抗性的关系。利用qPCR技术分析A03及C03染色体上P5CR同源基因在抗、感菌核病油菜材料中接种核盘菌前及接种后6 h、12 h、24 h、48 h的表达。结果 C03染色体上的P5CR同源基因全长1457 bp，该基因在抗、感菌核病油菜材料中共有7个SNP位点，其中3个SNP位点可能与抗病性相关；A03染色体上的P5CR同源基因全长1526 bp，在抗、感菌核病油菜材料中该基因共有15个SNP位点，其中2个SNP位点可能与抗病性相关。A03及C03染色体上的P5CR基因在抗病材料接种后24 h表达量显著升高。结论油菜P5CR基因上存在多个可能与菌核病抗性相关的位点且在抗病材料接种后表达升高，表明P5CR可能参与油菜对菌核病的抗性反应。本研究为进一步揭示甘蓝型油菜菌核病抗病机理及油菜抗菌核病分子标记开发奠定基础。
- P5CR /
- 菌核病 /
- 甘蓝型油菜 /
- 抗病性
Abstract: Objective The gene related to Sclerotinia-resistance in Brassica napus was cloned and studied to provide information for the development of disease-resistant rapeseed cultivars by means of molecular marker-assisted breeding. Method Using the rapeseed plants known to be either highly resistant or highly susceptible to sclerotinia stem rot, the homologous P5CR (pyrroline-5-carboxylate reductase) on A03 and C03 chromosomes were amplified, cloned, sequenced, and expression analyzed. DNAMAN software was used to compare the sequencing results to locate the relevant SNP sites. Expressions of P5CR before and 6 h, 12 h, 24 h, and 48 h after inoculation into rapeseed plants were detected by qPCR. Result The P5CR on C03 chromosome was 1457 bp in length with 7 SNP loci, of which, 3 might be related to the disease resistance. The gene on A03 chromosome was 1526 bp in length with 15 SNP sites, of which, two might be associated with the disease resistance. The expressions of P5CR on A03 and C03 chromosomes significantly increased 24 h after inoculation. Conclusion Multiple loci in the P5CR of B. napus could be associated with the plant resistance to sclerotinia stem rot. The significant increase on the gene expression after inoculation suggested a close relationship between P5CR and the disease resistance. Further investigation is needed to unveil the underline mechanism.
- P5CR /
- sclerotinia stem rot /
- Brassica napus /
- disease resistance

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图 1 甘蓝型油菜A03及C03染色体上P5CR同源基因扩增产物电泳图

A、B分别为A03及C03染色体上P5CR基因扩增电泳结果。Marker各条带分子量见图左侧标注。M，Marker III；1，川秦6R；2，南12R：3，中油821；4，沪16。

Figure 1. Electrophoretic map of amplified products of P5CR on chromosome A03 and C03 of B. napus

A & B: Amplified electrophoretic diagram of P5CRs on chromosome A03 and C03, respectively. Molecular weight of band of marker shown on left. M: Marker III; 1: Chuanqin 6R; 2: Nan 12R; 3: Zhongyou 821; 4: Hu 16.

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图 2 甘蓝型油菜A03染色体上P5CR基因与菌核病抗性相关的多态性位点

Figure 2. Polymorphic sites of P5CR on A03 chromosome of B. napus related to Sclerotinia-resistance

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图 3 甘蓝型油菜C03染色体上P5CR基因多态性分析

Figure 3. Polymorphism of P5CR on C03 chromosome of B. napus

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图 4 qPCR分析A03及C03染色体上P5CR基因在抗、感菌核病油菜材料中的表达结果

A、B分别为A03、C03染色体上P5CR基因的qPCR结果图。对同一时间不同材料中P5CR基因的表达量进行差异性分析（*表示P < 0.05，**表示P < 0.01）。

Figure 4. Expressions of P5CRs on A03 and C03 chromosomes in sclerotia-resistant and susceptible rapeseed materials detected by qPCR

A & B: qPCR data on P5CRs on A03 and C03 chromosomes, respectively. Expressions of P5CR gene in different materials at the same time (* indicates P < 0.05, ** indicates P < 0.01).

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表 1 油菜样品的编号、品种名称和表现型

Table 1. Codes, variety names, and phenotypes of B. napus samples

编号 Number	样本　　 Sample	抗性 Resistance
Y4	川秦6R Chuanqin 6R	S
Y7	南12R Nan 12R	S
Y8	中油821 Zhongyou 821	R
Y12	沪16 Hu 16	R

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表 2 甘蓝型油菜C03及A03染色体上P5CR同源基因引物序列信息

Table 2. Primer sequence of P5CR on chromosomes C03 and A03 of B. napus

引物名称 Primer name	序列（5′-3′） Sequence（5′-3′）	产物大小 Product length/bp
P5CRC03F2	GCCTTGGTAAGCGAATGG	1879
P5CRC03R2	TCGTACCCTGTGACGATTCA	1879
P5CRA03F1	TGTGTTGGGCCTTTGTAAAACAAT	1977
P5CRA03R1	TGCAATTTGGTCGTACCCTTTAAC	1977
qP5CR A03 F1	GGAAGTGGACCAGCATACG	197
qP5CR A03 R1	GCTATTGTAGTCCCGCCA	197
qP5CR C03 F1	TCGGAACAGCGGCAAGTGA	257
qP5CR C03R1	CTTCCCTGTCTTGCTCACCAT	257
Ubc21F2	TCCCGAACCGTATCCTCTGC	156
Ubc21R2	GGTTACCTGAGTCGCAGTTGAG	156

下载: 导出CSV

参考文献(21)

[1]	周颖. 中国冬油菜籽供给反应模型及实证分析[D]. 武汉: 华中农业大学, 2017. ZHOU Y. Study on the supply response model of Chinese winter rapeseed and its empirical analysis[D]. Wuhan: Huazhong Agricultural University, 2017. (in Chinese)
[2]	WEN L, TAN T L, SHU J B, et al. Using proteomic analysis to find the proteins involved in resistance against Sclerotinia sclerotiorum in adult Brassica napus [J]. European Journal of Plant Pathology, 2013, 137(3): 505−523. doi: 10.1007/s10658-013-0262-z
[3]	WANG Z R, WAN L L, ZHANG X H, et al. Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase: Implications for rapeseed resistance to fungal infection [J]. Planta, 2021, 253(2): 34. doi: 10.1007/s00425-020-03556-2
[4]	雷蕾, 梁龙兵, 秦信蓉, 等. 抗菌核病甘蓝型油菜种质的筛选与鉴定 [J]. 种子, 2020, 39(3):29−33. LEI L, LIANG L B, QIN X R, et al. Screening and identification of Sclerotinia-resistant germplasm of Brassica napus L [J]. Seed, 2020, 39(3): 29−33.(in Chinese)
[5]	WU J, CAI G Q, TU J Y, et al. Identification of QTLs for resistance to Sclerotinia stem rot and BnaC. IGMT5. a as a candidate gene of the major resistant QTL SRC6 in Brassica napus [J]. PLoS One, 2013, 8(7): e67740. doi: 10.1371/journal.pone.0067740
[6]	张羽, FRANCOIS BELZILE. 大豆抗菌核病的全基因组关联研究 [J]. 华北农学报, 2020, 35(1):205−213. doi: 10.7668/hbnxb.20190364 ZHANG Y, BELZILE F. Genome-wide association study for Sclerotinia sclerotiorum resistance of soybean [J]. Acta Agriculturae Boreali-Sinica, 2020, 35(1): 205−213.(in Chinese) doi: 10.7668/hbnxb.20190364
[7]	SENTHIL-KUMAR M, MYSORE K S. Ornithine-delta-aminotransferase and proline dehydrogenase genes play a role in non-host disease resistance by regulating pyrroline-5-carboxylate metabolism-induced hypersensitive response [J]. Plant, Cell & Environment, 2012, 35(7): 1329−1343.
[8]	周婉莹, 张晓娟, 孙晓敏, 等. 油菜P5CR基因克隆及其多态性分析 [J]. 基因组学与应用生物学, 2020, 39(12):5678−5683. doi: 10.13417/j.gab.039.005678 ZHOU W Y, ZHANG X J, SUN X M, et al. Cloning and polymorphism analysis of P5CR in rapeseed(B. napus) [J]. Genomics and Applied Biology, 2020, 39(12): 5678−5683.(in Chinese) doi: 10.13417/j.gab.039.005678
[9]	蓝碧秀, 王凛, 吴子恺, 等. 利用改良CTAB法快速小量提取微胚乳玉米基因组DNA [J]. 基因组学与应用生物学, 2015, 34(1):190−194. doi: 10.13417/j.gab.034.000190 LAN B X, WANG L, WU Z K, et al. Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method [J]. Genomics and Applied Biology, 2015, 34(1): 190−194.(in Chinese) doi: 10.13417/j.gab.034.000190
[10]	MEI J, QIAN L, DISI J O, et al. Identification of resistant sources against Sclerotinia sclerotiorum in Brassica species with emphasis on B. oleracea [J]. Euphytica, 2011, 177(3): 393−399. doi: 10.1007/s10681-010-0274-0
[11]	DING L N, LI M, GUO X J, et al. Arabidopsis GDSL1 overexpression enhances rapeseed Sclerotinia sclerotiorum resistance and the functional identification of its homolog in Brassica napus [J]. Plant Biotechnology Journal, 2020, 18(5): 1255−1270. doi: 10.1111/pbi.13289
[12]	LEBRETON S, CABASSA-HOURTON C, SAVOURÉ A, et al. Appropriate activity assays are crucial for the specific determination of proline dehydrogenase and pyrroline-5-carboxylate reductase activities [J]. Frontiers in Plant Science, 2020, 11: 602939. doi: 10.3389/fpls.2020.602939
[13]	FUNCK D, WINTER G, BAUMGARTEN L, et al. Requirement of proline synthesis during Arabidopsis reproductive development [J]. BMC Plant Biology, 2012, 12: 191. doi: 10.1186/1471-2229-12-191
[14]	DELAUNEY A J, VERMA D P. A soybean gene encoding delta 1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated [J]. Molecular & General Genetics:MGG, 1990, 221(3): 299−305.
[15]	CAO L, WEI S Q, HAN L, et al. Gene cloning and expression of the pyrroline-5-carboxylate reductase gene of perennial ryegrass (Lolium perenne) [J]. Horticultural Plant Journal, 2015, 1(2): 113−120.
[16]	SRIPINYOWANICH S, KLOMSAKUL P, BOONBURAPONG B, et al. Exogenous ABA induces salt tolerance in indica rice (Oryza sativa L.): The role of OsP₅CS₁ and OsP5CR gene expression during salt stress [J]. Environmental and Experimental Botany, 2013, 86: 94−105. doi: 10.1016/j.envexpbot.2010.01.009
[17]	王丽媛, 丁国华, 黎莉. 脯氨酸代谢的研究进展 [J]. 哈尔滨师范大学自然科学学报, 2010, 26(2):84−89. doi: 10.3969/j.issn.1000-5617.2010.02.024 WANG L Y, DING G H, LI L. Progress in synthesis and metabolism of proline [J]. Natural Science Journal of Harbin Normal University, 2010, 26(2): 84−89.(in Chinese) doi: 10.3969/j.issn.1000-5617.2010.02.024
[18]	MOLINARI H B C, MARUR C J, DAROS E, et al. Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): Osmotic adjustment, chlorophyll fluorescence and oxidative stress [J]. Physiologia Plantarum, 2007, 130(2): 218−229. doi: 10.1111/j.1399-3054.2007.00909.x
[19]	付莉莉, 韩冰莹, 谭德冠, 等. 木薯MeP5CS和MeP5CR基因克隆及其干旱胁迫下的表达分析 [J]. 湖北农业科学, 2016, 55(15):4024−4028. FU L L, HAN B Y, TAN D G, et al. Gene cloning of me P5CS and me P5CR in cassava and their expression analysis under drought stress [J]. Hubei Agricultural Sciences, 2016, 55(15): 4024−4028.(in Chinese)
[20]	XUE Y, PENG R H, XIONG A S, et al. Yeast heat-shock protein gene HSP26 enhances freezing tolerance in Arabidopsis [J]. Journal of Plant Physiology, 2009, 166(8): 844−850. doi: 10.1016/j.jplph.2008.11.013
[21]	DE RONDE J A, CRESS W A, KRÜGER G H J, et al. Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress [J]. Journal of Plant Physiology, 2004, 161(11): 1211−1224. doi: 10.1016/j.jplph.2004.01.014