水稻黑条矮缩病毒P7-1基因对水稻的遗传转化及转基因植株的抗病性分析

doi:10.16819/j.1001-7216.2018.8014

摘要/Abstract

摘要：

【目的】通过在水稻中过量表达水稻黑条矮缩病毒(Rice black streaked dwarf virus, RBSDV) P7-1基因,分析RBSDV P7-1是否是导致水稻不育的重要致病因子,并明确过量表达P7-1转基因植株对病毒的抗性。【方法】采用RT-PCR方法从感病植物中扩增获得RBSDV P7-1基因,构建pCAMBIA-1300-P7-1载体,通过农杆菌转化法获得转基因水稻,观察水稻是否能够正常结实,并采用人工接种病毒方法分析转基因植株的抗性。【结果】过量表达RBSDV P7-1的转基因水稻生长、结实正常。在接种7 d和14 d时转基因植株中病毒积累量显著低于野生型对照,但在28 d时部分转基因株系中病毒积累量高于对照,接种30 d时转基因植株的发病率与对照无明显差异。【结论】在水稻中过量表达RBSDV P7-1不影响水稻的结实;转基因水稻在RBSDV侵染早期可抑制病毒的积累,但在后期对病毒的抗性与对照没有明显差异。

关键词: 水稻黑条矮缩病毒, 过量表达, 育性, 抗性评价

Abstract:

【Objective】The purpose of this study is to determine whether Rice black streaked dwarf virus (RBSDV) P7-1 is an important pathogenic factor related to infertility in rice and to analyze the resistance of the P7-1 transgenic rice to RBSDV by overexpression of P7-1 in rice. 【Method】 RBSDV P7-1 gene was amplified from virus-infected plants by RT-PCR. The recombination plasmid pCAMBIA-1300-P7-1 was constructed and transformed to rice by Agrobacterium transformation. The fertility of transgenic plants was evaluated and the resistance of the transgenic rice plants to RBSDV was identified by artificial inoculation of virus. 【Result】The transgenic plants showed normal growth and fertility. The virus accumulation in the transgenic plants was significantly lower than the wild type at 7 days and 14 days after inoculation, but it was higher in some transgenic lines than the control at 28 days after inoculation. There were no significant differences in disease incidence between the transgenic plants and the wild type at 30 days after inoculation. 【Conclusion】 Overexpressing RBSDV P7-1 in rice does not affect the fertility. Transgenic rice had lower accumulation of virus in the early stage after RBSDV infection, but did not exhibit resistance to the virus at the later stage.

Key words: Rice black streaked dwarf virus, overexpression, fertility, resistance evaluation

中图分类号:

袁平平, 孙枫, 倪海平, 朱佳慧, 周益军, 蒋选利, 徐秋芳. 水稻黑条矮缩病毒P7-1基因对水稻的遗传转化及转基因植株的抗病性分析[J]. 中国水稻科学, 2018, 32(4): 342-348.

Pingping YUAN, Feng SUN, Haiping NI, Jiahui ZHU, Yijun ZHOU, Xuanli JIANG, Qiufang XU. Genetic Transformation of Rice black streaked dwarf virus P7-1 Gene in Rice and Its Virus Resistance Analysis[J]. Chinese Journal OF Rice Science, 2018, 32(4): 342-348.

图/表 8

表1 本研究所用引物

Table 1 Primers used in this study.

引物名称 Primer name	序列 Sequence(5′-3′)
P7-1-Asc I-F	GGCGCGCCATGGATAGACCTGCTCGAGAAC
P7-1-Pac I-R	TTAATTAAGCAGAAGGAGATGAAAAG
HPT-F	TTCCTTTGCCCTCGGACG
HPT-R	ACTCACCGCGACGTCTGT
P7-1-F1	ACCTCCTCCACCCCGAAA
P7-1-R1	GTGGAAGTGGAGATCGCCATA
OsActin-F	CAGCCACACTGTCCCCATCTA
OsActin-R	AGCAAGGTCGAGACGAAGGA
RBSDV P10-F	GCCCCACGTTGCATCTTC
RBSDV P10-R	TGTTGGGCAAAGTGCTAGTTTC

图1 pCAMBIA-1300-P7-1重组表达载体构建 A–PCR扩增RBSDV P7-1基因(M–DNA标记,泳道1和2分别为以健康和感病植株cDNA扩增P7-1的PCR产物);B–PCR筛选pMD18T-P7-1阳性克隆;C和D–pMD18-T-P7-1和pCAMBIA-1300-P7-1质粒的酶切分析(泳道5和7为重组质粒酶切产物,泳道6和8为未酶切的质粒对照)。

Fig. 1. Construction of the pCAMBIA-1300-P7-1 expression vector. A, PCR amplification of RBSDV P7-1 gene. M, DNA marker; Lane 1, cDNA of healthy rice; Lane 2, cDNA of virus-infected rice. B, Screening of positive clones of pMD18T-P7-1 by PCR; C and D, Digestion identification of recombinant plasmid pMD18T-P7-1 and pCAMBIA-1300-P7-1(Lanes 5 and 7, Digestion product; Lanes 6 and 8, Plasmid without enzyme digestion).

图2 T0转基因植株的PCR检测 M–DNA标记;1~52：转基因植株。

Fig. 2. PCR identification of T0 transgenic plants. M, DNA marker, Lanes 1 to 52, Transgenic rice lines.

图3 T0转基因植株P7-1和HPT基因的RT-PCR分析 A–RBSDV P7-1基因扩增; B–HPT基因扩增。M–DNA标记;1~52：转基因植株。

Fig. 3. Expression analysis of P7-1 and HPT gene in T0 transgenic plants by RT-PCR. A, RBSDV P7-1 amplification, B, HPT amplification. M, DNA marker, Lanes 1 to 52, Transgenic rice lines.

图4 RT-PCR检测T2转基因植株中P7-1的表达 M, DNA标记; #-14, #-19, #-40和#-46, T2转基因株系。下同。

Fig. 4. Detection of P7-1 genes in T2 plants by RT-PCR. M, DNA marker; #-14, #-19, #-40 and #-46, T2 transgenic plants. The same as below.

图5 qRT-PCR检测T2转基因植株中P7-1的表达柱上标不同小写字母代表差异达0.05显著水平。

Fig. 5. Expression of P7-1 gene in T2 transgenic plants by qRT-PCR. Different letters mean significant difference at 0.05 level.

图6 过量表达RBSDV P7-1 T2转基因植株的结实情况 WT, 野生型; #-14, #-19, #-40和#-46, T2转基因株系。下同。

Fig. 6. Seed setting of the T2 transgenic plants with overexpression of RBSDV P7-1. WT, Wild type; #-14, #-19, #-40 and #-46, T2 transgenic plants. The same below.

图7 转基因植株人工接种病毒不同时间后RBSDV P7-1表达量分析*代表与野生型相比差异达0.05显著水平。

Fig. 7. Relative expression of RBSDV P7-1 in transgenic plants as infected by virus for various time. *Compared with WT, difference was significant at 0.05 level.

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