杨树PagNAC47及其启动子克隆与功能分析

doi:10.16420/j.issn.0513-353x.2024-0674

摘要/Abstract

摘要：

以84K杨（Populus alba × Populus glandulosa）为试材，克隆得到PagNAC47基因及其启动子。PagNAC47编码区全长1 107 bp，编码368个氨基酸，具有NAC转录因子家族典型的NAM结构域，与毛果杨（P. trichocarpa）PtrNAC47蛋白的同源性最高，为92.2%；PagNAC47启动子序列长度为1 794 bp，含TATA-box和CAAT-box启动子基本元件及生长素（IAA）响应元件等。RT-qPCR分析表明，PagNAC47在老根、次生茎和成熟叶中的表达水平显著高于在幼根、初生茎和幼叶中的表达水平，且在成熟叶中表达水平最高。通过PagNAC47特异启动子驱动GUS报告基因的检测，揭示PagNAC47主要在器官的维管组织和成熟叶中表达。将35S启动子连接PagNAC47转入84K杨发现，与野生型相比，过表达株系的株高、茎粗、次生木质部宽度与层数、导管和纤维细胞面积及形成层层数均显著增加，根中次生木质部也有类似的现象，净光合速率和叶绿素含量显著增加，光合性能增强。PagNAC47的表达受IAA抑制，且抑制作用随IAA浓度的增加逐步增强。

关键词: 杨树, PagNAC47, 表达模式, 次生木质部, 光合作用, IAA

Abstract:

The PagNAC47 gene and its promoter were cloned from 84K poplar（Populus alba × Populus glandulosa）. Coding region of the PagNAC47 was 1 107 bp in length，encoding 368 amino acids，with a typical NAM domain of the NAC transcription factor family. It had the highest homology with the PtrNAC47 protein from P. trichocarpa，which was 92.2%. The promoter of PagNAC47 was 1 794 bp in length，containing the basic promoter elements of TATA-box and CAAT-box and auxin（IAA）response elements. RT-qPCR analysis showed that the expression level of PagNAC47 was significantly higher in old roots，secondary stems and mature leaves than that in young roots，primary stems and young leaves，and the expression level was the highest in mature leaves. Furthermore，detection of PagNAC47-specific promoter-driven GUS reporter gene revealed that the PagNAC47 was mainly expressed in vascular tissues of organs and mature leaves. The 35S promoter-linked PagNAC47 was transformed into 84K poplar，and it was found that transgenic poplar overexpressing PagNAC47 significantly increased plant height，stem thickness，secondary xylem width and layer，vessel and fiber cell area，and cambium layer compared with the wild type，and similar phenomena were also observed in the secondary xylem of roots. Compared with wild-type poplar，the net photosynthetic rate and chlorophyll content of overexpressing PagNAC47 84K poplar were significantly increased，and the photosynthetic performance was enhanced. The expression of PagNAC47 was inhibited by IAA，and the inhibitory effect gradually enhanced with the increase of IAA concentration.

Key words: poplar, PagNAC47, expression pattern, secondary xylem, photosynthesis, IAA

苏玉婷, 王慧林, 赵盼, 周心怡, 郑淑雅, 刘冉, 郭惠红. 杨树PagNAC47及其启动子克隆与功能分析[J]. 园艺学报, 2025, 52(7): 1828-1842.

SU Yuting, WANG Huilin, ZHAO Pan, ZHOU Xinyi, ZHENG Shuya, LIU Ran, GUO Huihong. Cloning and Functional Analysis of PagNAC47 Gene and Its Promoter from Poplar[J]. Acta Horticulturae Sinica, 2025, 52(7): 1828-1842.

图/表 15

表1 本研究中所用引物及序列

Table 1 The primers and sequences used in this study

用途 Application	引物名称 Primer name	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
基因克隆 Gene cloning	PagNAC47 F1/R1	ACCTCTAGGCTTCTTTCCCAGC	AATCAGACCCCCACACGCATC
	PagNAC47 F2/R2	ACGGGGGACTCTAGA GGATCCACCTCTAGGCTTCTTTCCCAGC	TATTCCCTGACTGGT GGGCCCAATCAGACCCCCACACGCATC
启动子克隆 Promoter amplification	proPagNAC47 F1/R1	GAACTGTTGATGAAAGGGAGGG	TTTGTCTGTTCCAGTTGCCTT
	proPagNAC47 F2/R2	GACCATGATTACGCC AAGC TTTACAGGCTTCATCCCAGC	ACCACCCGGGGATCC TCTAGAGCTAATCTAAGATTAGAA
转基因植株鉴定 Identification of transgenic plant	pBI-121F/R	CGTCTTCAAAGCAAGTGGATT	CCAACGCTGATCAATTCCAC
实时荧光定量PCR Fluorescent quantitative real-time	RT-qPCR F/R	GTTTAGGTTCCACCCGACAGA	CTCCCCCAAGGAAGATTTAGC
内参基因UBQ Reference gene UBQ	F/R	AGACCTACACCAAGCCCAAGAAGAT	CCAGCACCGCACTCAGCATTAG

图1 PagNAC47基因的克隆

Fig.1 Cloning of PagNAC47

图2 PagNAC47蛋白序列多重比对黑底代表几个物种的氨基酸相同。A、B、C、D和E代表保守基序。Pag：84K杨；Pal：银白杨；Ptr：毛果杨；At：拟南芥

Fig. 2 Multiple alignments of PagNAC47 protein sequences The black bottom represents that several species share the same amino acid. A、B、C、D and E represent conserved motif. Pag：Populus alba × Populus glandulosa；Pal：Populus alba；Ptr：Populus trichocarpa；At：Arabidopsis thaliana

图3 84K杨PagNAC47与其他物种NAC47蛋白进化树分析

Fig. 3 Phylogenetic analysis of 84K poplar PagNAC47 and NAC47 proteins from other plant species

图4 proPagNAC47的克隆

Fig. 4 Cloning of proPagNAC47

图5 PagNAC47基因的启动子序列及顺式作用元件 AuxRR-core：生长素响应元件；LTR：低温响应元件；ARE：厌氧诱导型元件；G-box：光反应的顺式作用元件；TGACG-motif：茉莉酸甲酯响应元件

Fig. 5 PagNAC47 gene promoter sequence and cis-acting elements AuxRR-core：Auxin response element；LTR：Cryogenic responsive elements；ARE：Anaerobic-inducing elements；G-box：cis-Acting element of light reaction；TGACG-motif：Methyl jasmonate response element

图6 84K杨PagNAC47基因的表达模式分析不同的小写字母表示显著性差异（P < 0.05）

Fig. 6 Expression pattern analysis of PagNAC47 in 84K poplar Different lowercase letters indicate significant differences（P < 0.05）

图7 proPagNAC47转基因拟南芥的PCR鉴定

Fig. 7 PCR identification of proPagNAC47 transgenic Arabidopsis thaliana

图8 proPagNAC47::GUS转基因拟南芥GUS检测

Fig. 8 GUS detection of proPagNAC47::GUS transgenic Arabidopsis

图9 PagNAC47转基因杨树的PCR（A）、GUS（B）检测和RT-qPCR分析（C） 1：阴性对照；2：假阳性植株；3 ~ 5：转基因阳性植株。不同的小写字母表示显著性差异（P < 0.05）

Fig. 9 PCR（A），GUS detection（B）and RT-qPCR analysis（C）of PagNAC47 transgenic poplar 1：Negative control；2：False positive plants；3-5：Transgenic positive plants.Different lowercase letters indicate significant differences（P < 0.05）

图10 过表达PagNAC47 84K杨与野生型84K杨数据统计不同的小写字母表示显著性差异（P < 0.05）

Fig. 10 Statistics of overexpressed PagNAC47 84K poplar and wild-type 84K poplar Different lowercase letters indicate significant differences（P < 0.05）

图11 过表达PagNAC47 84K杨（OE）与野生型84K杨的横切面细胞图 Xy：次生木质部；Ca：形成层；Ph：韧皮部；Ve：导管；Xf：木纤维

Fig. 11 Cross-sectional cell diagramof overexpressed PagNAC47 84K poplar（OE）and wild-type 84K poplar Xy：Xylem；Ca：Cambium；Ph：Phloem；Ve：Vessel；Xf：Xylary fiber

图12 过表达PagNAC47 84K杨（OE）与野生型84K杨（WT）的数据统计不同的小写字母表示显著性差异（P < 0.05）

Fig. 12 Statistics of overexpressed PagNAC47 84K poplar（OE）and wild-type 84K poplar（WT） Different lowercase letters indicate significant differences（P < 0.05）

图13 过表达PagNAC47 84K杨与野生型84K杨的光合指标分析不同的小写字母表示显著性差异（P < 0.05）

Fig. 13 Analysis of photosynthetic indexes of overexpressed PagNAC47 84K poplar and wild-type 84K poplar Different lowercase letters indicate significant differences（P < 0.05）

图14 外源IAA处理对84K杨PagNAC47表达量的影响不同的小写字母表示显著性差异（P < 0.05）

Fig. 14 The effect of exogenous IAA treatment on the expression level of PagNAC47 in 84K poplar Different lowercase letters indicate significant differences（P < 0.05）

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