中国农业科学 ›› 2021, Vol. 54 ›› Issue (5): 877-886.doi: 10.3864/j.issn.0578-1752.2021.05.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

过量表达OsRRK1对水稻叶片发育的影响

马银花1(),莫凯琴1,刘璐1,李萍芳1,金晨钟1(),杨芳2()   

  1. 1湖南人文科技学院农业与生物技术学院/湖南省农田杂草防控技术与应用协同创新中心,湖南娄底 417000
    2武汉大学生命科学学院/杂交水稻国家重点实验室,武汉 430072
  • 收稿日期:2020-07-16 接受日期:2020-08-26 出版日期:2021-03-01 发布日期:2021-03-09
  • 通讯作者: 金晨钟,杨芳
  • 作者简介:马银花,E-mail:mayinhua1988@126.com
  • 基金资助:
    国家自然科学基金(31800201);湖南省自然科学基金青年项目(2019JJ50281);湖南省教育厅优秀青年项目(18B455);杂交水稻国家重点实验室(武汉大学)开放课题基金(KF201901);娄底市应用技术与开发项目(33319013);湖南省应用特色学科(植物保护)

Effect of Overexpression of OsRRK1 Gene on Rice Leaf Development

YinHua MA1(),KaiQin MO1,Lu LIU1,PingFang LI1,ChenZhong JIN1(),Fang YANG2()   

  1. 1School of Agriculture and Biotechnology, Hunan University of Humanities, Science and Technology/Hunan Provincial Collaborative Innovation Center for Field Weeds Control, Loudi 417000, Hunan
    2College of Life Sciences, Wuhan University/National Key Laboratory of Hybrid Rice, Wuhan 430072
  • Received:2020-07-16 Accepted:2020-08-26 Online:2021-03-01 Published:2021-03-09
  • Contact: ChenZhong JIN,Fang YANG

摘要:

【目的】OsRRK1(rop-interacting receptor-like kinase 1)属于胞质类受体激酶RLCK Ⅵ家族成员,通过对OE-OsRRK1转基因水稻的叶片形态观察,明确OsRRK1在水稻叶片发育中的作用。【方法】依据水稻基因组数据库公布的目标序列设计引物并构建OsRRK1超量表达载体,利用农杆菌介导的遗传转化转入到粳稻品种合江19,通过PCR分析鉴定阳性植株,Southern blot鉴定转基因植株插入外源基因的拷贝数,Northern blot杂交和qRT-PCR鉴定转基因植株在RNA水平的表达情况;选取T2代纯合植株抽穗期的剑叶进行叶片卷曲指数(leaf rolling index,LRI)测定;通过石蜡切片和苯胺蓝染色观察水稻横切后泡状细胞的变化情况,用Image J软件统计泡状细胞的面积;采用叶绿素测定计测量植株叶片叶绿素含量。【结果】共获得OE-OsRRK1转基因阳性植株44株,随机选取17株,经Southern blot鉴定8株为单拷贝株系,9株为多拷贝株系;随机选取2份多拷贝株系(OE-1和OE-4)和5份单拷贝株系(OE-5、OE-21、OE-22、OE-24和OE-25)用于后续试验。经分析发现OsRRK1在不同的转基因株系中具有不同程度的过表达情况,其中OE-1、OE-4和OE-25株系超量表达程度高,OE-5株系超量表达程度低,OE-21、OE-22和OE-24株系超量表达程度居中。从中选取OE-5、OE-22和OE-25共3份单拷贝转基因株系和WT对照经Northern blot鉴定后与qRT-PCR结果一致。统计选取的7个株系和WT对照的叶片卷曲指数发现:卷曲程度与OsRRK1表达量呈正相关,表达量越高卷曲程度越高。水稻叶片经切片和染色后发现OE-OsRRK1转基因植株叶片中泡状细胞发生了明显变化,转基因株系中泡状细胞数目都小于对照中泡状细胞均值4.6个。叶片卷曲得越明显,泡状细胞退化得越严重,数目更少,面积更小。经叶绿素含量测定发现,OE-22、OE-24和OE-25转基因的水稻叶片的叶绿素含量比野生型水稻叶片叶绿素含量高。【结论】过量表达OsRRK1会影响泡状细胞的数目和面积,从而使水稻叶片发生卷曲,且卷曲程度与表达量呈正相关;OsRRK1在水稻中的过量表达导致叶绿素含量增高。

关键词: 水稻, OsRRK1, 胞质类受体激酶, 叶片卷曲, 叶绿素含量

Abstract:

【Objective】OsRRK1 (RoP-Interacting receptor-like kinase 1) is a member of the cytoplasmic receptor kinases RLCK VI family. In this study, the leaf morphology of OE-OsRRK1 transgenic rice was observed to clarify the role of OsRRK1 in the leaf development of rice. 【Method】Primers were designed based on target sequences published in the rice genome database to construct OsRRK1 overexpression vector, then transformed to Japonica variety Hejiang 19 mediated by Agrobacterium tumefaciens. Positive plants were identified by PCR analysis. The copy number of inserted exogenous genes in transgenic plants was identified by Southern blot hybridization. The expression level of transgenic plants at RNA level was confirmed by Northern blot hybridization and qRT-PCR. T2 generation homozygote plants at heading stage was selected to determine the leaf rolling index (LRI). Paraffin sections and aniline blue staining were used to observe the changes of rice bulliform cells after cross-cutting, and Image J software was used to calculate the area of the bubble cells. Chlorophyll content in plant leaves was measured by chlorophyll tester.【Result】44 positive transgenic plants were obtained and 17 lines were randomly selected for Southern blot analysis. Among the 17 lines, 8 lines were single-copy insertion and 9 lines were multi-copy insertion. 2 multi-copy lines (OE-1 and OE-4) and 5 single-copy lines (OE-5, OE-21, OE-22, OE-24 and OE-25) were randomly selected for subsequent analysis. qRT-PCR analysis revealed that OsRRK1 was overexpressed with different degree in different transgenic lines. Among them, OE-1, OE-4 and OE-25 lines with the highest OsRRK1 expression level, while OE-5 lines with the lowest and OE-21, 22 and 24 lines with the middle. Three single copy transgenic lines OE-5, OE-22 and OE-25 and WT control were selected for Northern blot analysis. The results were in consistent with the results of qRT-PCR results. Leaf rolling index of the seven selected lines and WT control revealed that the degree of leaf curl was positive correlated with the expression level of OsRRK1, with the higher the expression level, the higher the degree of rolling. Paraffin section and staining revealed the number and area of bulliform cells in the flag leaves of OE-OsRRK1 transgenic plants changed obviously compared to WT. OE-OsRRK1 transgenic plants showed less bulliform cell number than those in the control whose average is 4.6. OE-OsRRK1 transgenic plants with curlier leaf, with the more severe bulliform cell degeneration, less number, smaller area. Chlorophyll content determination revealed that the OE-22, OE-24 and OE-25 lines have higher chlorophyll content than those of the WT rice leaves.【Conclusion】The overexpression of OsRRK1affect the number and size of bulliform cells in rice leaves, which cause leaf rolling, and the degree of leaf rolling is positively correlated with the expression level of OsRRK1. The overexpression of OsRRK1 gene will lead to the increase of chlorophyll content in rice.

Key words: Oryza sativa, OsRRK1, cytoplasmic receptor-like kinase, leaf rolling, chlorophyll content