茶树CsNPF6.1/6.3基因克隆及ABA转运功能验证

doi:10.16420/j.issn.0513-353x.2023-0768

摘要/Abstract

摘要：

为探索茶树（Camellia sinensis）NPF转运蛋白的激素转运特性，以‘龙井43’叶片为模板，克隆获得2个完整的茶树NPF基因CsNPF6.1和CsNPF6.3，其CDS序列长度分别为1 665和1 788 bp，分别编码554和595个氨基酸。系统发育树分析表明，CsNPF6.1和CsNPF6.3与狭叶油茶（Camellia lanceoleosa）的ClNPF相似性最高，分别是98.19%和97.82%。在线预测CsNPF6.1和CsNPF6.3亚细胞定位均位于细胞质膜，为典型的膜转运蛋白。组织表达分析显示，CsNPF6.1和CsNPF6.3在茶树嫩叶、成熟叶和根中均有表达，其中嫩叶最高。在ABA处理条件下，CsNPF6.1和CsNPF6.3在嫩叶中表达量受诱导显著降低，根中则显著升高。经ABA依赖的酵母双杂交体系鉴定，在100 μmol · L^-1 ABA处理下CsNPF6.1和CsNPF6.3均可促进转基因酵母的生长，且可显著提高酵母细胞内ABA的含量。综上，茶树中CsNPF6.1和CsNPF6.3均具有转运ABA的能力。

关键词: 茶树, CsNPF6.1, CsNPF6.3, 基因克隆, ABA转运

Abstract:

To explore the hormone transport characteristics of tea（Camellia sinensis）NPF transporter，two complete NPF genes（CsNPF6.1 and CsNPF6.3）were cloned from‘Longjing 43’leaves. The CDS（coding sequence）of CsNPF6.1 and CsNPF6.3 were 1 665 bp and 1 788 bp encoding 554 and 595 amino acids，respectively. Phylogenetic analysis showed that CsNPF6.1 and CsNPF6.3 had the highest identical sequence identity with ClNPF（Camellia lanceoleosa），which were 98.19% and 97.82% respectively. Both CsNPF6.1 and CsNPF6.3 were predicted to be located in the plasma membrane and were typical membrane transporters. Tissue-specific expressions revealed that they were expressed in tender leaves，mature leaves，and roots，and mainly accumulated in tender leaves. Under ABA treatment，the expression levels of CsNPF6.1 and CsNPF6.3 were significantly decreased in tender leaves，but significantly increased in roots. According to the identification of ABA-dependent yeast two-hybrid system，CsNPF6.1 and CsNPF6.3 could promote the growth of transgenic yeast cells and could significantly increase the content of ABA in yeast cells under 100 μmol · L^-1 ABA condition. In conclusion，both CsNPF6.1 and CsNPF6.3 in tea plants have the function to transport ABA.

Key words: tea plant, CsNPF6.1, CsNPF6.3, gene cloning, ABA transport

袁青云, 韩昱, 贺巍, 苏会, 班秋艳, 吴春来, 周琼琼, 徐文静, 王丽鸳, 张芬. 茶树CsNPF6.1/6.3基因克隆及ABA转运功能验证[J]. 园艺学报, 2024, 51(12): 2817-2828.

YUAN Qingyun, HAN Yu, HE Wei, SU Hui, BAN Qiuyan, WU Chunlai, ZHOU Qiongqiong, XU Wenjing, WANG Liyuan, ZHANG Fen. Cloning and ABA Transport Function Analysis of CsNPF6.1/6.3 in Tea Plants[J]. Acta Horticulturae Sinica, 2024, 51(12): 2817-2828.

图/表 10

表1 基因克隆、表达以及酵母表达载体构建引物

Table 1 Primers sequence of gene cloning，expression and the yeast expression vector construction

引物用途 Purpose	引物名称 Primers name	引物序列（5′-3′） Primer sequence
基因克隆 Gene clone	CsNPF6.1 CsNPF6.3	F：ATACCGCCGCTGGAACTACA；R：GAAGTGCTAGCTTTCATAGGGGG F：CATTCATTCAATACGACCCAACTAC；R：TCTTTATGCATGGCAAGTAGGC
基因表达 Gene expression	GAPDH-q CsNPF6.1-q CsNPF6.3-q	F：TTGGCATCGTTGAGGGTCT；R：CAGTGGGAACACGGAAAGC F：CGATAGCAGCGTGTTTGTGG；R：GGCTGCCTTGTCCAAACATC F：GCCTCACTGACTGTTTTCTTCG；R：AACCGCTTGATTTCGCCTA
载体构建 Vectors construction	AtHAB1-NdeⅠ AtHAB1-SmaⅠ AtPYR1-NdeⅠ AtPYR1-SmaⅠ	F：gtaccagattacgct catatgCATATGGTATGGAGGAGATGACTCC； R：ccgtatcgatgccca cccgggCCCGGGTCAGGTTCT GGTCTTGAAC F：tcagaggaggacctg catatgATGCCTTCGGAGTTAACACCAG； R：gcaggtcgacggatc cccgggTCACGTCACCTGAGA ACCACTTC

图1 茶树CsNPF6.1和CsNPF6.3基因克隆

Fig. 1 Gene cloning of CsNPF6.1 and CsNPF6.3 in tea plants

图2 茶树CsNPF6.1和CsNPF6.3氨基酸序列对比黑色背景代表高度相似的氨基酸序列信息。

Fig. 2 The amino acid sequence comparison of CsNPF6.1 and CsNPF6.3 in tea plants Black boxes indicate conserved amino acid residues.

图3 茶树CsNPF6.1和CsNPF6.3蛋白的系统发育和保守基序颜色方块代表目的基因的10个保守基序。

Fig. 3 Phylogenetic and conserved motif of CsNPF6.1 and CsNPF6.3 proteins in tea plants Different colors represent the 10 conserved motifs of the target genes.

图4 茶树CsNPF6.1和CsNPF6.3基因组织特性分析

Fig. 4 Organizational characteristics analysis of CsNPF6.1 and CsNPF6.3 genes in tea plants ** P < 0.01.

图5 ABA处理后‘龙井43’和‘中茶108’茶树CsNPF6.1和CsNPF6.3在不同组织部位的表达水平

Fig. 5 The expression levels of CsNPF6.1 and CsNPF6.3 in different tissues of‘Longjing 43’and‘Zhongcha 108’in tea plants after ABA treatment * P < 0.05，** P < 0.01.

图6 AD-HAB1/BD-PYR1体系阳性克隆验证

Fig. 6 Positive clones of the AD-HAB1/BD-PYR1 system

图7 AD-HAB1/BD-PYR1体系自激活验证 1-1、1-2、1-3为3次重复。

Fig. 7 Self-activation verification of AD-HAB1/BD-PYR1 system 1-1，1-2 and 1-3 are three repetitions.

图8 AD-HAB1/BD-PYR1体系CsNPF6.1和CsNPF6.3激素转运检测

Fig. 8 Detection of hormone transport activities of CsNPF6.1 and CsNPF6.3 proteins using AD-HABI/BD-PYR1 system

图9 转基因酵母细胞ABA转运特性分析

Fig. 9 The analysis of ABA transport characteristics of transgenic yeast cells * P < 0.05.

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