水稻干尖线虫Ab-lea基因在高渗透压下的表达

doi:10.16819/j.1001-7216.2017.7004

摘要/Abstract

摘要：

【目的】水稻干尖线虫(Aphelenchoides besseyi)在侵染寄主以前,能够以变渗隐生状态在土壤和种子中的高渗透压环境下长期存活,使防治该线虫极为困难。通常,非生物胁迫下,胚胎发育晚期丰富蛋白(late embryogenesis abundant protein,LEA)的基因表达量会显著上调。作为逆境表达蛋白,LEA能在逆境下保护生物。为探究水稻干尖线虫LEA编码基因(Ab-lea)在高渗透压胁迫下的表达模式及功能,【方法】根据转录组数据克隆到Ab-lea。以无机溶剂饱和MgSO₄·7H₂O溶液为高渗透压胁迫剂,对水稻干尖线虫进行高渗透压胁迫,240 min后该线虫进入变渗隐生。通过RT-PCR验证Ab-lea在线虫进入变渗隐生及维持变渗隐生过程中的表达特性。使用RNAi技术,探究该线虫Ab-lea沉默后,高渗透压胁迫下存活率变化。【结果】 RT-PCR结果表明,Ab-lea在进入变渗隐生前期及维持变渗隐生过程中表达量上调。Ab-lea沉默后,水稻干尖线虫在进入变渗隐生后存活率显著下降。【结论】表明该基因可能参与调控水稻干尖线虫变渗隐生。本研究为进一步探究该线虫抗高渗透压胁迫的生理机制奠定基础,为防治该线虫提供新思路。

关键词: 胚胎发育晚期丰富蛋白, 水稻干尖线虫, 高渗透压胁迫, 变渗隐生, 基因干涉

Abstract:

【Objective】Rice white tip nematodes (Aphelenchoides besseyi) can enter osmobiosis to survive hypertonic osmotic stress in soil and seeds before infecting hosts, making the prevention and control of the nematode rather difficult. Normally, under hypertonic osmotic stress, the gene expression of late embryogenesis abundant protein (LEA) will significantly increase. As an adversity expressive protein, LEA can protect the organism under adversity. In order to study the expression pattern and the function of LEA gene from A. besseyi (Ab-lea) under hypertonic osmotic stress, 【Method】the gene was cloned according to the results of the transcriptome sequencing. A saturated MgSO₄·7H₂O solution was used as a hypertonic osmotic solution to dehydrate nematodes. The expression of Ab-lea was tested by RT-PCR during Osmobiosis. RNAi was used to investigate the survival of nematodes under hypertonic osmotic stress after silencing of Ablea.【Result】Nematodes will turn into osmobiosis after soaking in a saturated MgSO₄·7H₂O solution for 240 min. RT-PCR results showed that the expression of Ab-lea was upregulated when nematodes were at the beginning of hypertonic osmotic stress treatment and during the osmobiosis. 【Conclusion】The survival rate of the nematodes dropped significantly under osmobiosis after Ab-lea silence, which showed that Ab-lea may be involved in the regulation of osmobiosis of the nematode. It lays a foundation of further exploration for the physiological mechanism of nematode-resistance to hypertonic osmotic stress and provides new ideas for the prevention and control of the nematode.

Key words: late embryogenesis abundant protein, Aphelenchoides besseyi, hypertonic osmotic stress, osmobiosis, RNAi

中图分类号:

S435.111.4⁺8

陈俏丽, 王峰, 李丹蕾, 零雅茗, 张瑞芝. 水稻干尖线虫Ab-lea基因在高渗透压下的表达[J]. 中国水稻科学, 2017, 31(6): 652-657.

Qiaoli CHEN, Feng WANG, Danlei LI, Yaming LING, Ruizhi ZHANG. Expression Under Hypertonic Osmotic Stress of Ab-lea from Aphelenchoides besseyi[J]. Chinese Journal OF Rice Science, 2017, 31(6): 652-657.

图/表 6

表1 NCBI登录的胚胎发育晚期丰富蛋白(LEA)直系同源序列

Table 1 Late embrgogenesis abundant protein(LEA) orthologous sequence in NCBI.

物种 Species	GenBank登录号 NCBI No.	蛋白 Protein	E 值 E value
鼠类圆线虫 Strongyloides ratti	CEF69463	假定蛋白SRAE_2000411200 Hypothetical protein SRAE_2000411200	6×10^-11
秀丽隐杆线虫Caenorhabditis elegans	NP_001256172.1	植物胚胎发育晚期丰富蛋白相关 Plant LEA related	3×10^-9
广杆属线虫Caenorhabditis brenneri	EGT57645.1	假定蛋白CAEBREN_08606 Hypothetical protein CAEBREN_08606	5×10^-9
广杆属线虫Caenorhabditis remanei	XP_003116340.1	CRE-LEA-1蛋白 Protein CRE-LEA-1	4×10^-8
广杆属线虫Caenorhabditis briggsae	CAP25449.2	CBR-LEA-1蛋白 Protein CBR-LEA-1	1×10^-7
犬蛔虫Toxocara canis	KHN75743.1	假定蛋白Tcan_16220 Hypothetical protein Tcan_16220	4×10^-6

图1 水稻干尖线虫RNA(A)、Cdna(B)和Ab-lea ORF(C)扩增

Fig. 1. RNA(A), cDNA(B) and the Ab-lea (C)PCR product of A. besseyi.

图2 高渗透压胁迫处理后水稻干尖线虫体长变化柱上横线表示标准差。*,**分别表示在0.05,0.01水平上差异显著。下同。

Fig. 2. Changes in length of A. besseyi during hypertonic osmotic stress treatment. Error lines are stardand deviation.*,** indicate significant difference at 0.05 and 0.01 levels, respectively. The same as in figures below.

图3 高渗透压胁迫处理后水稻干尖线虫体型变化 A,B,C,D,E,F,G,H,I,J分别代表在饱和MgSO4·7H2O溶液中浸泡0,10,20,30,40,50,60,120,180,240min。比例尺为100 μm。

Fig. 3. The changes of morphology of A. besseyi during hypertonic osmotic stress treatment. A, B, C, D, E, F, G, H, I, J indicate A.beseyi is soaked in saturated MgSO4·7H2O solution for 0, 10, 20, 30, 40, 50, 60, 120, 180, 240 min, respectively. Bar= 100 µm.

图4 高渗透压胁迫处理后水稻干尖线虫Ab-lea基因表达量情况

Fig. 4. Expression of Ab-lea during hypertonic osmotic stress treatments.

图5 Ab-lea基因沉默后高渗透压胁迫处理水稻干尖线虫存活数

Fig. 5. Survival of A. besseyi under hypertonic osmotic stress after Ab-lea RNAi.

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