谷子类受体蛋白激酶基因SiRLK35在水稻中对盐胁迫的响应

doi:10.3864/j.issn.0578-1752.2019.22.004

中国农业科学 ›› 2019, Vol. 52 ›› Issue (22): 3976-3986.doi: 10.3864/j.issn.0578-1752.2019.22.004

谷子类受体蛋白激酶基因SiRLK35在水稻中对盐胁迫的响应

李小波^1,²,李臻²,戴绍军³,潘教文²,王庆国²,管延安^4,⁵,丁国华¹(),刘炜^2,⁵()

1 哈尔滨师范大学生命科学与技术学院,哈尔滨 150025
2 山东省农业科学院生物技术研究中心/山东省作物遗传改良与生理生态重点实验室,济南 250100
3 上海师范大学生命与环境科学学院/植物种质资源开发协同创新中心,上海 200234
4 山东省农业科学院作物研究所/山东省特色作物工程实验室,济南 250100
5 山东师范大学生命科学学院,济南 250014

收稿日期:2019-07-02 接受日期:2019-09-19 出版日期:2019-11-16 发布日期:2019-11-16
通讯作者: 丁国华,刘炜
作者简介:李小波,E-mail：1670065759@qq.com
基金资助:
现代农业产业技术体系建设专项(CARS-06-13.5-A19);山东省农业科学院农业科技创新工程(CXGC2018E13);山东省农业科学院青年英才计划(2016—2018)

Response of Receptor-Like Protein Kinase Gene SiRLK35 of Foxtail Millet to Salt in Heterologous Transgenic Rice

LI XiaoBo^1,²,LI Zhen²,DAI ShaoJun³,PAN JiaoWen²,WANG QingGuo²,GUAN YanAn^4,⁵,DING GuoHua¹(),LIU Wei^2,⁵()

1 School of Life Sciences and Technology, Harbin Normal University, Harbin 150025
2 Biotechnology Research Center, Shandong Academy of Agricultural Sciences/Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan 250100
3 College of Life and Environmental Sciences, Shanghai Normal University/Development Center of Plant Germplasm Resources, Shanghai 200234
4 Crop Research Institute, Shandong Academy of Agricultural Sciences/Shandong Engineering Laboratory for Featured Crops, Jinan 250100
5 College of Life Sciences, Shandong Normal University, Jinan 250014

Received:2019-07-02 Accepted:2019-09-19 Online:2019-11-16 Published:2019-11-16
Contact: GuoHua DING,Wei LIU

摘要/Abstract

摘要：

【目的】盐胁迫影响作物的产量和品质,而作物的耐盐性受特定基因的调控。在前期获得谷子类受体蛋白激酶基因SiRLK35过表达水稻株系的基础上,拟结合植株在盐胁迫下的表型,对部分盐胁迫指标及响应基因的表达模式进行检测和验证,解析谷子SiRLK35参与盐害响应的可能机制。【方法】选取谷子SiRLK35过表达水稻株系,分别为过表达株系（over-expression,OE）-1（OE-1）、OE-2和OE-3,以野生型中花11为对照,实时荧光定量PCR（quantitative real-time PCR,qRT-PCR）检测各株系中SiRLK35的表达情况;分别用含0、150和200 mmol·L ^-1 NaCl的MS培养液处理三叶期的对照及转基因株系幼苗,观察其表型,统计150 mmol·L ^-1 NaCl处理3 d后苗长及根长;用150 mmol·L ^-1 NaCl处理四叶期幼苗,统计处理14 d后材料干重、死亡率和死叶率,对各材料的耐盐性进行评价;进一步挑选SiRLK35过表达程度高且耐盐表型好的OE-1株系,利用3,3'-二氨基联苯胺（3,3'-Diaminobenzidine,DAB)和氮蓝四唑（nitrotetrazolium blue chloride,NBT）染色检测其胁迫下过氧化物积累及超氧化物歧化酶（superoxide dismutase,SOD）和过氧化物酶（peroxidase,POD）的活性;并对部分盐害响应基因的表达模式进行检测。【结果】谷子SiRLK35在株系OE-1中的相对表达量最高;150 mmol·L ^-1NaCl处理3 d幼苗后,中花11苗长和根长的生长受抑制程度均大于SiRLK35过表达株系,其中OE-1的受抑制程度最小;四叶期幼苗经150 mmol·L ^-1 NaCl处理14 d后,转基因株系地上部和地下部干重降低幅度均低于对照,且幼苗死亡率和死叶率均低于对照;盐胁迫下对照过氧化染色反应明显,O ^2-和H₂O₂的积累均高于过表达植株,SOD和POD抗氧化酶活性均高于对照;部分盐害响应基因在SiRLK35过表达株系中表现为上调,其中,OsLEA3在盐处理24 h的相对表达量是对照中的1.9倍。【结论】谷子SiRLK35异源转化水稻获得的过表达株系对盐胁迫具有一定的抗性,SiRLK35可通过调控抗氧化酶活性及相关信号途径,从而参与盐害响应。

关键词: 谷子, SiRLK35, 耐盐性, 抗氧化酶, 盐胁迫响应基因

Abstract:

【Objective】Salt stress affects the crop yield and quality, while the crop salt tolerance is regulated by specific genes. Using heterogeneous over-expressed rice lines (OE-1, OE-2, and OE-3) of foxtail millet receptor-like protein kinase gene SiRLK35, the possible mechanisms of SiRLK35 under salinity will be dissected on phenotypic, physiological and molecular levels. 【Method】 The expressions of SiRLK35 in OE-1, OE-2, and OE-3 were analyzed by qRT-PCR. The phenotypes of three-leaf stage seedlings treated with 0, 150 and 200 mmol·L ^-1 NaCl were observed, and the lengths of seedlings and roots were measured after treated with 150 mmol·L ^-1 NaCl for 3 days. The dry weights, death rate and dead leaf rate of four-leaf stage seedlings that treated with 150 mmol·L ^-1 NaCl for 14 days were also measured. OE-1with the highest SiRLK35 expression was further used for DAB and NBT dyeing analysis. The activities of partial antioxidases, and expression patterns of marker genes were detected. 【Result】 There was the highest SiRLK35 expression level in OE-1. The growth of control and OE seedlings were all inhibited under 150 mmol·L ^-1 NaCl. The decreased levels of dry weight, the death rate and dead leaf rate of OE rice were all lower than those of the control, together with the less accumulation of O ^2- and H₂O₂, and the higher activities of antioxidases under salinity. Partial of salt responsive genes were up-regulated in SiRLK35 OE lines, especially the OsLEA3 was induced about 1.9 times higher after treated with NaCl for 24 h. 【Conclusion】 SiRLK35 OE rice lines have tolerance under salinity, and the gene SiRLK35 of foxtail millet could participate in salt response by regulation the activities of antioxidases and related signal pathways.

Key words: foxtail millet (Setaria italica L.), SiRLK35, salt tolerance, antioxidase, salt responsive genes

李小波,李臻,戴绍军,潘教文,王庆国,管延安,丁国华,刘炜. 谷子类受体蛋白激酶基因SiRLK35在水稻中对盐胁迫的响应[J]. 中国农业科学, 2019, 52(22): 3976-3986.

LI XiaoBo,LI Zhen,DAI ShaoJun,PAN JiaoWen,WANG QingGuo,GUAN YanAn,DING GuoHua,LIU Wei. Response of Receptor-Like Protein Kinase Gene SiRLK35 of Foxtail Millet to Salt in Heterologous Transgenic Rice[J]. Scientia Agricultura Sinica, 2019, 52(22): 3976-3986.

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参考文献 38

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基因名称Gene name	登录号Accession number	正向引物Forward primer (5'-3')	反向引物Reverse primer (5'-3')
OsACTIN	MS971743.1	TCGGCTACAACCCTGACAA	CAAACTTGACGGCAATGTGG
SiRLK35	XM_004956247.2	AAATAGTTGGGTAGACGAGG	TACGCAGATGATTGACAGAT
OsP5CS	D49714.1	CTATTCCTCGTAATGTTGG	CTTAGTTGACGCCTTGAT
OsLEA3	Z68090.1	ACAAGGACACCTCTGCCACC	AATAGACCCAAAGGGAAATCA
OsNHX1	AB021878.1	GAGAGGAGCTGTGTCGATTG	CTCTCGAGGTCAGAACCTTG
OsHKT1	KU501218.1	ACCATAAGCACAACCCAG	AATCCTAAGAACCACCTCA
OsNAC6	AB028185.1	GATCATGCACGAGTACCGC	GCACCCAATCATCCAACCT
OsDREB2A	JQ341059.1	GCTGAGATCCGTGAACCA	ACCATACATTGCCCTTGC

谷子类受体蛋白激酶基因SiRLK35在水稻中对盐胁迫的响应

Response of Receptor-Like Protein Kinase Gene SiRLK35 of Foxtail Millet to Salt in Heterologous Transgenic Rice

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