烟草NtNRAMP3b的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0382

摘要/Abstract

摘要：

铁影响植物的生长发育，在动植物中，天然抗性相关巨噬蛋白NRAMP（natural resistance-associated macrophage protein）家族主要负责Fe、Mn和Cd等金属离子的吸收与转运，研究NtNRAMP3b对Fe的转运机制，为明确NtNRAMP3b在烟草转运铁中的机理提供理论基础。以烟草品种K326的cDNA为模板，克隆烟草NtNRAMP3b，并分析该基因的序列特征，利用RT-qPCR测定NtNRAMP3b的表达模式，运用转基因技术获得NtNRAMP3b-OE（NtNRAMP3b overexpression），并测定其铁浓度。NtNRAMP3b全长1 530 bp、编码509个氨基酸序列。NtNRAMP3b与AtNRAMP3的亲缘关系最近，同源性达74.85%。RT-qPCR结果表明，NtNRAMP3b在叶片中表达量最高，缺铁处理后的相对表达量均增加。缺铁处理后，与野生型相比，NtNRAMP3b-OE生物量增加，叶绿素浓度增加，铁含量降低。NtNRAMP3b主要参与铁元素从液泡向细胞器转运，提高了叶片中铁的吸收与再利用。

关键词: 烟草, NtNRAMP3b, 缺铁, 铁含量

Abstract:

Iron（Fe）affects plant growth and development，and NRAMP（natural resistance-associated macrophage protein）in animals and plants is mainly responsible for the absorption and transport of metals ions such as Fe，Mn and Cd. Studying the transport mechanism of NtNRAMP3b to Fe is to provide a theoretical basis for clarifying the mechanism of NtNRAMP3b in tobacco（Nicotiana tabacum）transport iron. Taking the cDNA of tobacco cultivar K326 as a template，the tobacco’s NtNRAMP3b was cloned and its sequence characteristics were analyzed，the instantaneous expression of NtNRAMP3b was determined by RT-qPCR. Using transgenic technology to obtain NtNRAMP3b-OE（NtNRAMP3b overexpression），and its iron concentration was determined. NtNRAMP3b was 1 530 bp long and encoded 509 amino acid sequences. NtNRAMP3b was most closely related to AtNRAMP3，with 74.85% homogeneity. RT-qPCR results showed that NtNRAMP3b was expressed the most in leaves，and the relative expression after iron deficiency treatment increased. Compared with the wild type，NtNRAMP3b-OE biomass increased，chlorophyll concentration increased，and iron content decreased after iron deficiency treatment. NtNRAMP3b is mainly involved in the transport of iron from vacuoles to organelles，improving the absorption and reuse of iron in leaves.

Key words: Nicotiana tabacum, NtNRAMP3b, iron deficiency, iron content

尹卓然, 轩栋栋, 李晨依, 李长, 柴哲, 王锟瑶, 赵孟琦, 彭靖媛, 董杰, 贾宏昉. 烟草NtNRAMP3b的克隆及功能分析[J]. 生物技术通报, 2022, 38(12): 175-183.

YIN Zhuo-ran, XUAN Dong-dong, LI Chen-yi, LI Chang, CHAI Zhe, WANG Kun-yao, ZHAO Meng-qi, PENG Jing-yuan, DONG Jie, JIA Hong-fang. Cloning and Functional Analysis of Gene NtNRAMP3b in Nicotiana tabacum[J]. Biotechnology Bulletin, 2022, 38(12): 175-183.

图/表 7

图1 NtNRAMP3b跨膜结构域分析

Fig. 1 Cross-membrane domain analysis of NtNRAMP3b

图2 同源性分析

Fig. 2 Homology analysis

图3 系统进化树和motif分析

Fig. 3 Phylogenetic tree and motif analysis

图4 启动子顺式元件分析

Fig. 4 Promoter cis-element analysis

图5 NtNRAMP3b相对表达量不同字母表示在P<0.01水平差异极显著

Fig. 5 NtNRAMP3b relative expression Different letters indicate significant differences in the P< 0.01 level

图6 不同铁浓度处理下的生物量在同一处理下，对WT、OE1与OE2进行差异显著性分析，*表示在P<0.05水平差异显著；**表示在P<0.01水平差异极显著。下同

Fig. 6 Biomass under treatment with different cadmium concentrations In the same treatment, *and ** indicate OE1 and OE2 has a significant difference in the P<0.05 or P< 0.01 compared to WT. The same below

图7 缺铁条件下基因的瞬时表达

Fig. 7 Transient expressions of genes under iron deficiency

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