Cloning and ABA Transport Function Analysis of CsNPF6.1/6.3 in Tea Plants

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

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (12): 2817-2828.doi: 10.16420/j.issn.0513-353x.2023-0768

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Cloning and ABA Transport Function Analysis of CsNPF6.1/6.3 in Tea Plants

YUAN Qingyun¹^,², HAN Yu¹, HE Wei¹, SU Hui¹, BAN Qiuyan¹, WU Chunlai¹, ZHOU Qiongqiong¹, XU Wenjing¹, WANG Liyuan²(), ZHANG Fen¹()

¹College of Horticulture，Henan Agricultural University，Zhengzhou 450046，China
²Tea Research Institute，Chinese Academy of Agricultural Sciences，Hangzhou 310008，China

Received:2024-07-13 Revised:2024-09-25 Online:2024-12-25 Published:2024-12-13
Contact: WANG Liyuan, ZHANG Fen

Abstract

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

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.

Figures/Tables 10

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

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

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

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.

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

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.

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

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

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

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

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Cloning and ABA Transport Function Analysis of CsNPF6.1/6.3 in Tea Plants

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