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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)

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
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