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作物学报 ›› 2021, Vol. 47 ›› Issue (6): 1043-1053.doi: 10.3724/SP.J.1006.2021.04069

• 专题：主要麻类作物基因组学与遗传改良 • 上一篇    下一篇

利用红麻HcPDIL5-2a非全长基因创制雄性不育新种质

周步进1,2(), 李刚3, 金刚4, 周瑞阳1,*(), 刘冬梅5, 汤丹峰3, 廖小芳4, 刘一丁1, 赵艳红4, 王颐宁1   

  1. 1广西大学农学院, 广西南宁 530004
    2广西农业科学院玉米研究所, 广西南宁 530007
    3广西药用植物园, 广西南宁 530023
    4广西农业科学院经济作物研究所, 广西南宁 530007
    5商丘师范学院生物与食品学院, 河南商丘 476000
  • 收稿日期:2020-03-17 接受日期:2021-01-13 出版日期:2021-06-12 网络出版日期:2021-01-22
  • 通讯作者: 周瑞阳
  • 作者简介:E-mail:zhou_bujin@163.com
  • 基金资助:
    国家自然科学基金项目(31571719);广西自然科学基金项目(2018JJB130072)

Creation of male sterile germplasm using the partial length gene of HcPDIL5-2a in kenaf

ZHOU Bu-Jin1,2(), LI Gang3, JIN Gang4, ZHOU Rui-Yang1,*(), LIU Dong-Mei5, TANG Dan-Feng3, LIAO Xiao-Fang4, LIU Yi-Ding1, ZHAO Yan-Hong4, WANG Yi-Ning1   

  1. 1College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China
    2Maize Research Institute of Guangxi Academy of Agricultural Sciences, Nanning 530007, Guangxi, China
    3Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, Guangxi, China
    4Cash Crop Institute of Guangxi Academy of Agricultural Sciences, Nanning 530007, Guangxi, China
    5Shangqiu Normal University, Shangqiu 476000, Henan, China
  • Received:2020-03-17 Accepted:2021-01-13 Published:2021-06-12 Published online:2021-01-22
  • Contact: ZHOU Rui-Yang
  • Supported by:
    The National Natural Science Foundation of China(31571719);The Natural Science Foundation of Guangxi(2018JJB130072)

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摘要：

为创造转基因雄性不育种质, 将缺失酶活性中心序列的红麻HcPDIL5-2a非全长基因通过花粉管通道法转化红麻保持系722B, 获得了海南冬繁不育、南宁夏繁可育的低温敏雄性不育种质722THS, 并从其姊妹交后代选育出了稳定型核不育(GMS)系722HS; 此后, 又以722HS作母本, 以非转基因野生型722B为轮回亲本连续回交, 选育出了细胞质雄性不育系722HA。对722THS与722HA的细胞学观察和线粒体DNA分子鉴定表明, 二者的小孢子败育时期均为双核期, 但722HA发生了线粒体DNA重排, 而722THS与722B的线粒体DNA保持不变。本研究结果为转基因创造作物雄性不育种质找到了一条新途径, 具有重要的科学意义和广阔的应用前景。

关键词: 红麻, 雄性不育, HcPDIL5-2a非全长基因

Abstract:

In order to create transgenic male sterile germplasm in kenaf, the partial length of gene HcPDIL-2a was transformed into maintainer line 722HB via pollen tube pathway. A low-temperature sensitive male sterile mutant called ‘722THS’ was accquired, which showed sterile when grown in winter in Hainan and fertile when grown in summer in Nanning, and a stable Genic Male Sterile (GMS) line 722HS was selected from its sister progenies. A cytoplasmic male sterile line 722HA was bred by crossing the mutant 722THS with the wild type 722B. Cytological observation indicated that the microspore abortion of 722THS and 722HA both happened at dinuclear stage. The mitochondrial DNA molecular tag revealed that mitochondrial DNA rearrangement occurred in 722HA, while mitochondrial DNA of 722THS and 722B remained unchanged. This study provided a new way for the creation of male sterile germplasms, which had important scientific significances and broad application prospects.

Key words: kenaf, male sterility, partial length HcPDIL-2a

图1

PBI121-PH-GFP 质粒载体图谱"

图2

红麻雄性不育突变体722THS与野生型722B及其CMS系722A花的形态特征"

图3

红麻突变体722THS及野生型722B小孢子发育观察"

表1

红麻722THS/722B杂交F1和F2育性统计"

杂交组合
Cross		 可育株数
Number of fertile plants		 不育株数
Number of sterile plants
722THS-1/722B 86 0
722THS-2/722B 113 0
(722THS-1/722B)自交 (722THS-1/722B) selfing 130 0
(722THS-2/722B)自交 (722THS-2/722B) selfing 95 0

图4

722THS自然杂交F1代不育株PCR检测 M: DNA marker; 1: 阳性质粒; 2: 阴性对照; 3~6: 不育株。"

图5

722THS环割可育自交后代NPT II检测(广西南宁) M: DNA marker; 1~6为不育株; 7~31为可育株。"

表2

722THS海南环割可育自交后代育性统计(海南)"

统计时间
Count date		 平均气温
Average
temperature (℃)		 杂交组合
Hybrid combination		 可育株数
Number of fertile plants		 不育株数
Number of sterile plants		 期望比例
Expect proportion		 χ2 显著性测验
Significance test
χ2 (0.05,1)
开花初期
(3月14-16日)
Early flowering (from March 14
to 16)		 23.3 722THS-S1环割可育自交
722THS-S1 girdling fertile selfing		 123 91 3:1 35.05 NS
722THS-S2环割可育自交722THS-S2 girdling fertile selfing 263 193 3:1 72.99 NS
722THS-S3环割可育自交722THS-S3 girdling fertile selfing 133 101 3:1 41.17 NS
722THS-S4环割可育自交722THS-S4 girdling fertile selfing 17 44 3:1 61.55 NS
开花后期
(4月11-13日)
Late flowering
(from April 11
to 13)		 27.5 722THS-S1环割可育自交
722THS-S1 girdling fertile selfing		 158 56 3:1 0.24 *
722THS-S2环割可育自交722THS-S2 girdling fertile selfing 324 122 3:1 1.50 *
722THS-S3环割可育自交722THS-S3 girdling fertile selfing 163 71 3:1 3.56 *
722THS-S4环割可育自交722THS-S4 girdling fertile selfing 43 18 3:1 0.66 *

图6

红麻722HA、722HB花的形态特征[23]"

图7

红麻722HA及722HB小孢子发育观察[23] PMC: 花粉母细胞; Td: 四分体小孢子; Ms: 单核期小孢子; Ds: 双核期小孢子; MP: 成熟花粉粒; Ep: 外皮层; En: 内皮层; ML: 中间层; T: 绒毡层; 比例尺为20 μm。"

图8

分子标签MM509、MM556检测不同材料的胞质类型 M: DNA marker; 1~4分别为722A、722HA、722B和722THS。"

图9

分子标签MM509、MM556在不同材料中的序列比对 A: 分子标签MM509序列比对结果; B: 分子标签MM556序列比对结果。"

表3

红麻CMS系722HA、722A与恢复系杂交后代育性统计"

组合
Cross		 可育株数
Number of fertile plants		 不育株数
Number of sterile plants
722A/福红992 722A/Fuhong 992 56 0
722A/R7 91 0
722HA/福红992 722HA/Fuhong 992 52 14
722HA/R7 44 35
[1] 史春霞, 白洋, 郁崇文. 黄、红麻资源的优化与开发利用. 中国麻作, 2001,24(1):40-44.
Shi C X, Bai Y, Yu C W. Optimization and development of jute and kenaf resources. China’s Fiber Crops, 2001,24(1):40-44 (in Chinese).
[2] 程舟, 鲛岛一彦, 陈家宽. 日本的红麻研究、加工和利用. 中国麻业, 2001,23(3):16-24.
Cheng Z, Samejima K, Chen J K. Kenaf research, processing and utilization in Japan. Plant Fiber Prod, 2001,23(3):16-24 (in Chinese)
[3] 陶爱芬, 张晓琛, 祁建民. 红麻综合利用研究进展与产业化前景. 中国麻业科学, 2007,29(1):2-6.
Tao A F, Zhang X C, Qi J M. Research progress and industrialization prospect of the comprehensive utilization in kenaf. Plant Fiber Sci China, 2007,29(1):2-6 (in Chinese).
[4] Srivastava S K, Pandey B P B, Lal R S. Combing ability in a six-parent diallel cross in Mesta. Ind J Agric Sci, 1978,49:724-740.
[5] Pace S, Piscioneri I, Settanni I. Heterosis and combining ability in a half diallel cross of kenaf (Hibiscus cannabinus L.) in south Italy. Ind Crops Prod, 1998,7:417-427.
[6] Ugale S D, Khuspe S S. Cytoplasmic genetic male sterility in Hibiscus cannabinus L. J Maharashtra Agric Univ, 1976,1:102-106.
[7] 汤永海, 李德芳. 红麻杂种二代利用的研究和探讨. 中国麻作, 1991, (3):4-9.
Tang Y H, Li D F. Study on the utilization of hybrids second generation in kenaf. China’s Fiber Crops, 1991, ( 3):4-9 (in Chinese).
[8] 周瑞阳, 张新, 张加强, 甘正华, 韦汉西. 红麻细胞质雄性不育系的选育及杂种优势利用取得突破. 中国农业科学, 2008,41:314.
Zhou R Y, Zhang X, Zhang J Q, Gan Z H, Wei H X. A breakthrough in kenaf cytoplasmic male sterile lines breeding and heterosis utilization. Sci Agric Sin, 2008,41:314 (in Chinese with English abstract).
[9] 李德芳, 陈安国, 唐慧娟. 红麻质核互作型雄性不育系的发现及初步创制. 中国麻业科学, 2007,29(2):78.
Li D F, Chen A G, Tang H J. Discovery and creation of cytoplasmic and nuclear benetic male sterility in kenaf (Hibiscus cannabinus L.). Plant Fiber Sci China, 2007,29(2):78 (in Chinese).
[10] Zhao Y H, Chen P, Liao X F, Zhou B J, Liao J, Huang Z P, Kong X J, Zhou R Y. A comparative study of the atp9 gene between a cytoplasmic male sterile line and its maintainer line and further development of a molecular marker specific for male sterile cytoplasm in kenaf(Hibiscus cannabinus L.). Mol Breed, 2014,42:969-976.
[11] Zhao Y H, Liao X F, Zhou B J, Zhao H T, Zhou Y Y, Zhou R Y. Mutation in the coding sequence of atp6 are associated with male sterile cytoplasm in kenaf (Hibiscus cannabinus L.). Euphytica, 2016,207:169-175.
[12] 陈鹏, 周琼, 周瑞阳, 赵艳红, 廖小芳, 周步进, 何冰. 利用红麻ZB90的不育细胞质选育雄性不育系的方法. 中国专利: ZL 201310710624.8. 2013-12-19.
Chen P, Zhou Q, Zhou R Y, Zhao Y H, Liao X F, Zhou B J, He B. The method of breeding male sterile line using ZB90 sterile cytoplasm in kenaf. Chinese patent: ZL 201310710624.8. 2013-12-19 (in Chinese).
[13] 赵艳红, 廖小芳, 周瑞阳, 陈鹏, 周琼, 周步进. 利用红麻 KN250 的不育细胞质选育雄性不育系的方法 中国专利: ZL 201310714202.8 2013-12-19.
Zhao Y H, Liao X F, Zhou R Y, Chen P, Zhou Q, Zhou B J. The method of breeding male sterile line using KN250 sterile cytoplasm in kenaf. Chinese patent: ZL 201310714202.8. 2013-12-19 (in Chinese).
[14] 廖小芳, 赵艳红, 周瑞阳, 陈鹏, 周琼, 周步进. 利用红麻 KN142 的不育细胞质选育雄性不育系的方法 中国专利: ZL 201310711047.4. 2013-12-19.
Liao X F, Zhao Y H, Zhou R Y, Chen P, Zhou Q, Zhou B J. The method of breeding male sterile line using KN142 sterile cytoplasm in kenaf. Chinese patent: ZL 201310711047.4. 2013-12-19 (in Chinese).
[15] Ferrari D M, Soling H D. The protein disulphide-isomerase family: unravelling a string of folds. Biochem J, 1999,339:1-10.
[16] Freedman R B, Hirst T R, Tuite M F. Protein disulphide isomerase: building bridges in protein folding. Trends Biochem Sci, 1994,19:331-336.
pmid: 7940678
[17] Han X H, Wang Y H, Liu X, Jiang L, Ren Y L, Liu F, Peng C, Li J J, Jin X M, Wu F Q. The failure to express a protein disul-phide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice. J Exp Bot, 2012,63:121-130.
[18] Wang X, Chen J, Liu C A, Luo J L, Yan X, Ai A H, Cai Y H, Xie H W, Ding X, Peng X J. Over-expression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice. Gene, 2019,684:124-130.
[19] 耿威. 蛋白二硫键异构酶基因沉默和超表达对水稻高温结实和品质性状影响. 浙江大学硕士学位论文,浙江杭州, 2015.
Geng W. Influence of High Temperature on Rice Pikelet Fertility and Grain Quality in Relation to Protein Disulfide Isomerase (PDI) Gene Silencing and Over-expression. MS Thesis of Zhejiang University, Hangzhou, Zhejiang,China, 2015 (in Chinese with English abstract).
[20] Yang K Z, Xia C, Liu X L, Dou X Y, Wang W, Chen L Q, Zhang X Q, Xie L F, He L Y, Ma X, Ye D. A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis. Plant J, 2009,57:870-882.
[21] 李刚. 红麻细胞质雄性不育系线粒体蛋白质组差异分析与不育相关基因的发掘. 广西大学博士学位论文,广西南宁, 2009.
Li G. Analysis of Mitochondrial Proteomic Differeces and Discovery of Related Genes in Cytoplasmic Male Sterility in Kenaf (Hibiscus cannabinus L.). PhD Dissertation of Guangxi University, Nanning, Guangxi,China, 2009 (in Chinese with English abstract).
[22] 金刚. 红麻CMS胞质的SNP标记转化及不育系和保持系表达差异的研究. 广西大学博士学位论文广西,南宁, 2011.
Jin G. Conversion of SNP Markers Associated with CMS Cytoplasm and Differential Expression Analysis in Anther of CMS and Its Maintainer Line of Kenaf (Hibiscus cannabinus L.). PhD Dissertation of Guangxi University, Nanning, Guangxi,China, 2011 (in Chinese with English abstract).
[23] Zhou B J, Liu Y D, Chen Z X, Liu D M, Wang Y N, Zheng J, Liao X F, Zhou R Y. Comparative transcriptome analysis reveals the cause for accumulation of reactive oxygen species during pollen abortion in cytoplasmic male-sterile kenaf line 722HA. Int J Mol Sci, 2019,20:5515.
[24] Kroczynska B, Blond S Y. Cloning and characterization of a new soluble murine J-domain protein that stimulates BiP, Hsc70 and DnaK ATPase activity with different efficiencies. Gene, 2001,273:267-274.
[25] Xu S, Ding H, Sang J R. Reactive oxygen species, metabolism, and signal transduction in plant cells. Acta Bot Yunnanica, 2007,29:355-365.
[26] Wallace D C. Mitochondrial DNA mutations in disease and aging. Environ Mol Mutagen, 2010,51:440-450.
[27] Kumar M, Pathak D, Kriplani A, Ammini A C, Talwar P, Dada R. Nucleotide variations in mitochondrial DNA and supra- physiological ROS levels in cytogenetically normal cases of premature ovarian insufficiency. Arch Gynecol Obstet, 2010,282:695-705.
[28] Venkatesh S, Deecaraman M, Kumar R, Shamsi M B, Dada R. Role of reactive oxygen species in the pathogenesis of mitochondrial DNA (mtDNA) mutations in male infertility. Indian J Med Res, 2009,129:127-137.
[29] 周瑞阳, 刘冬梅. 棉花转基因创制细胞质雄性不育系的方法. 中国专利: ZL 201710061030.7. 2017-01-25.
Zhou R Y, Liu D M. The method of creating cytoplasmic male sterile line by transgene in cotton. Chinese Patent: ZL 201710061030.7. 2017-01-25 (in Chinese).
[30] 周瑞阳, 汤丹峰. 水稻转基因创制细胞质雄性不育系的方法. 中国专利: ZL 201710061028.X. 2017-01-25.
Zhou R Y, Tang D F. The method of creating cytoplasmic male sterile line by transgene in rice. Chinese Patent: ZL 201710061028.X. 2017-01-25 (in Chinese).
[31] 范树国, 梁承邺, 刘鸿先. 离体条件下核不育水稻突变为细胞质雄性不育. 植物学报, 2000,17:548-554.
Fan S G, Liang C Y, Liu H X. In vitro mutation of rice from GMS to CMS. Chin Bull Bot, 2000,17:548-554 (in Chinese with English abstract).
[32] 牛景, 牛芝霞. 光敏核不育水稻杂种F2花药培养中获得了细胞质不育突变体. 杂交水稻, 2001,16(2):42.
Niu J, Niu Z X. A cytoplasmic male sterile mutant was obtained from the anther culture of the F2 of a cross of photoperiod sensitive genic male sterile rice. Hybrid Rice, 2001,16(2):42 (in Chinese with English abstract).
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