<i>Rpfan37</i>在刺槐共生结瘤过程中的功能探究 <sup>*</sup>

doi:10.13523/j.cb.20180507

中国生物工程杂志

2018, Vol. 38

Issue (5): 47-55 DOI: 10.13523/j.cb.20180507

研究报告

Rpfan37在刺槐共生结瘤过程中的功能探究 ^*

冯昭^1,²,丑敏霞^2,^**(

)

1 陕西中医药大学医学技术学院咸阳 712046
2 西北农林科技大学旱区作物逆境生物学国家重点实验室杨陵 712100

Functional Analysis of Rpfan37 in the Symbiotic Nodulation Process of Robinia Pseudoacacia

Zhao FENG^1,²,Min-xia CHOU^2,^**(

)

1 College of Medical Technology, Shaanxi University of Chinese Medicine, Xianyang 712046, China
2 State Key Laboratory of Crop Stress Biology in Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China;

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摘要：

目的:研究刺槐中与磷脂酰肌醇转运蛋白有较高同源性的基因Rpfan37的功能,为探究相关基因参与豆科植物与根瘤菌共生结瘤过程提供新的思路。方法:通过前期研究,建立豆科植物刺槐与共生根瘤菌互作的抑制差减杂交反交文库,筛选疑似与共生结瘤相关的基因。利用PCR技术快速克隆经实时荧光定量PCR技术(qRT-PCR)分析基因在不同接菌时间及不同植物组织的表达。构建RNA干扰(RNAi)重组载体,转农杆菌介导转化植物根部,接种根瘤菌后验证该基因在刺槐共生结瘤过程的功能。结果:基因表达分析显示,在接菌与未接菌的刺槐根中,处理后第15天,Rpfan37表达均显著上调,但接菌与未接菌处理对该基因表达无显著影响;在成熟的根瘤中,该基因仅为低水平表达。RNAi转化植株的鲜重、株高、根长及结瘤数较对照组显著降低。在显微镜下观察到RNAi植株根毛发育异常;与对照相比,RNAi转化植株形成的根毛卷曲、根毛侵染线及根瘤原基数目均显著降低。根瘤石蜡切片结果显示RNAi植株根瘤中的侵染细胞与对照相比明显减少,分析豆血红蛋白表达发现,RNAi植株中根瘤发育成熟过程明显受阻。结论:在豆科植物刺槐中发现的相关基因Rpfan37能够参与刺槐共生结瘤过程,为研究磷脂酰肌醇转运蛋白在共生结瘤过程中的作用提供了新的理论基础。

关键词： Rpfan37; 共生结瘤; RNA干扰; 磷脂酰肌醇转运蛋白; 刺槐

Abstract:

Objective:To investigate the new gene Rpfan37, a homolog of PITP, in order to provide an idea for understanding the function of relative genes that involved in the symbiotic nodulation process.Methods:Through the previous study, target genes which were suspectly associated with symbiotic nodulation were screened out from the suppression substractive hybridization library of Robinia pseudoacacia interacting with symbiotic rhizobia. Using qRT-PCR to analyze the target gene expression level at different times and in different tissues. The RNAi recombinant plasmid was transformed into the plant root through K599 and verify the function of Rpfan37 after inoculation.Results:qRT-PCR analysis showed up-regulation characteristics of Rpfan37 in roots especially at 15 days post-inoculation (dpi), however, inoculation and non-inoculation treatment had no significant effect on the gene expression which sharply decreased in the matured nodule. Knockdown of Rpfan37 via RNA interference resulted in impaired development of both plant growth and nodule. Compared with empty vector plants, fresh weight, the root and stem length, nodule number per plant deceased dramatically in Rpfan37 RNAi plants. The root hair development of RNAi plant was abnormal and the number of root hair curling, ITs and nodule primordia were also significantly reduced in the Rpfan37 RNAi roots. Nodulation paraffin sections showed that the number of infected cells in RNAi plant nodules was significantly reduced compared with the control. Real-time PCR analysis of the expression levels of leghemoglobin gene indicated that nodule development and maturation was significantly blocked in the Rpfan37 RNAi roots.Conclusion:The related gene Rpfan37 found in Robinia pseudoacacia can participate in the symbiotic nodulation process. It provides a new theoretical basis for understanding the function of phosphatidylinositol transporter in symbiotic nodulation process.

Key words: Rpfan37 Symbiotic nodulation RNA interference Phosphatidylinositol transfer protein Robinia pseudoacacia

收稿日期: 2018-02-02 出版日期: 2018-06-05

ZTFLH:

Q945

基金资助: * 国家自然科学基金资助项目(31172252);* 国家自然科学基金资助项目(31370142)

通讯作者: 丑敏霞 E-mail: minxia95@163.com

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引用本文:

冯昭,丑敏霞. Rpfan37在刺槐共生结瘤过程中的功能探究 ^*[J]. 中国生物工程杂志, 2018, 38(5): 47-55.

Zhao FENG,Min-xia CHOU. Functional Analysis of Rpfan37 in the Symbiotic Nodulation Process of Robinia Pseudoacacia. China Biotechnology, 2018, 38(5): 47-55.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180507 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I5/47

表1 本研究中所用的引物

图1 Rpfan37在不同时期根与根瘤中的相对表达量

图2 RNAi植株GUS染色及GUS基因和目的基因在RNAi植株中的表达水平检测

图3 Rpfan37干扰后对刺槐的生长表型、根瘤形态及结瘤数的影响

图4 接菌后8d的对照和RNAi根毛及侵染线形态

图5 Rpfan37 RNAi对植株根毛卷曲、侵染线及根瘤原基数目的影响

图6 接菌后30天刺槐根瘤纵切光学显微镜照片

图7 接菌后15d和30d刺槐根瘤豆血红蛋白基因表达分析

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