《植物生理学报》 2021, 57(4): 879-889

转ChIFN基因烟草的TMV抗性与生长及光合特性

肖芳斌¹，董亚萍¹，张冬生¹，宋莉^1,*，赵德刚^1,2,*

¹贵州大学生命科学学院, 农业生物工程研究院, 山地植物资源保护与种质创新教育部重点实验室, 贵州省农业 生物工程重点实验室, 贵阳550025；²贵州省农业科学院, 贵阳550006

通信作者：宋莉；E-mail: lpsssl@126.com；赵德刚；E-mail: dgzhao@gzu.edu.cn

摘　要：

烟草感染烟草普通花叶病毒(TMV)后可引起叶片畸形并出现花斑枯斑, 严重影响烟草品质。前期将具有抗病毒和细胞生长调节等广泛生物学活性的鸡干扰素糖蛋白编码基因(ChIFN)遗传转化烟草进行TMV抗性研究。对获得的T2代ChIFN-α、 ChIFN-γ以及它们的杂交F₁代ChIFN-αγ三种转基因烟草(TP)进行鉴定, 并分析TP烟草与其野生型亲本(WT)之间的TMV抗性及生长发育差异。结果表明, 外源ChIFN基因在T₂代和F₁代TP烟草中稳定表达, 以TP_R5、 TP_G4和TP_RG2植株中的重组干扰素(rChIFN)累积量最高, 分别为rChIFN-α 27.05 ng·L^–1、 rChIFN-γ 29.52 ng·L^–1、 rChIFN-αγ 52.64 ng·L^–1。 TMV侵染离体培养叶片24 h时,三种TP烟草的病毒量分别比WT烟草低14.87%、 8.72%和14.36%。 TMV接种后, TP_R5 、 TP_G4、 TP_RG2烟草的同期叶绿素a和叶绿素总量降幅均低于WT, 12 h时叶绿素b的保持能力为TP_RG2>TP_G4>TP_R5>WT, 0~24 h时三种TP烟草叶片的类胡萝卜素降幅均低于同期WT。 TMV侵染处理的三种TP烟草发芽率分别比WT高36.67%、50.00%、 52.00%, 发芽势分别高19.59%、 28.00%、 31.33%。转ChIFN基因烟草对TMV具有良好的抗性。种子萌发实验表明TP烟草种子萌发提前约5 d。三种TP烟草25 d移栽幼苗的株高分别比WT增加5.18%、 23.29%和44.71%, 具有株高生长优势; TP和WT型烟草的叶绿素、净光合速率、气孔导度、蒸腾速率等光合参数无显著差异。研究结果可为抗TMV病毒的转ChIFN基因烟草应用提供依据。

关键词： 鸡干扰素; 转基因烟草; 烟草花叶病毒; 抗病性; 发芽率; 发芽势

收稿：2020-10-20   修定：2021-03-17

资助：国家转基因生物新品种培育科技重大专项项目(2016ZX08010003)、国家自然科学基金(31660685)和贵州省科技计划项目(黔科合 基础[2017]1044号、黔科合平台人才[2017]5788号)。

TMV resistance, growth and photosynthetic characteristics of transgenic tobacco with ChIFN genes

XIAO Fangbin¹, DONG Yaping¹, ZHANG Dongsheng¹, SONG Li^1,*, ZHAO Degang^1,2,*

¹College of Life Sciences and Institute of Agro-Bioengineering, Guizhou University, Key Laboratory of Plant Resources Conservationand Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou Key Lab of AgroBioengineering, Guiyang 550025, China 2; ²Guizhou Academy of Agricultural Sciences, Guiyang 550006, China

Corresponding author: SONG Li; E-mail: lpsssl@126.com; ZHAO Degang; E-mail: dgzhao@gzu.edu.cn

Abstract:

Tobacco mosaic virus (TMV) can seriously affect tobacco quality by causing the leaf deformities
and variegated spots. ChIFN genes encoding chicken alpha interferon and gamma interferon which have a
wide range of biological activities such as antiviral and cell growth regulation were transformed to tobacco plants in previous study. In this study, the transgenic tobacco (TP) of T₂ generation of ChIFN-α, ChIFN-γ and their F₁ hybrid were analysed on the TMV resistance, growth and developmentcompared to their wild-type parent (WT). Results showed the highest expression level were rChIFN-α 27.05 ng·L^-1, rChIFN-γ 29.52 ng·L^-1 and rChIFN-αγ 52.64 ng·L^-1 in 3 kinds of TP tobacco respectively. The virus in 3 TP leaves were 14.87%、8.87%和14.36% lower than WT after TMV inoculation 24 h. Total decrease of chlorophyll a and chlorophyll content in TP leaves were lower than WT at the same time. The chlorophyll b level was TP_RG2>TP_G4>TP_R5>WT in different leaves at 12 h. The degradation of carotenoids in TP_RG2, TP_G4 and TP_R5 were all lower than WT during 0-24 h. Transgenic tobacco expressing ChIFN genes has well TMV resistance than WT. The germination rate of 3 TP seed treated with TMV was 36.67%, 50.00%, 52.00% higher than WT. And the germination vigor was 19.59%, 28.00%, 31.33% higher than WT. It was observed that the seed germination was 5 days earlier than WT. The plant height of 25 days TP seedlings were increased 5.18%, 23.29% and 44.71% respectively than WT in TP_R5, TPG4 and TPRG2 lines. However, the photosynthetic performance index of chlorophyll, net photosynthetic rate, stomatal conductance and transpiration rate have no signifcant difference between TP and WT lines. These results can provide a strong evidence for the application of transgenic ChIFN tobacco.

Key words: chicken interferon; transgenic tobacco; Tobacco mosaic virus; virus resistance; germination rate; germination vigor