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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (01): 40-51.doi: 10.3724/SP.J.1006.2019.94066

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and functional analysis of promoter of potassium transporter gene GhHAK5 in upland cotton (Gossypium hirsutum L.)

Mao-Ni CHAO1,Hai-Yan HU1,*(),Run-Hao WANG1,Yu CHEN2,Li-Na FU1,Qing-Qing LIU1,Qing-Lian WANG1   

  1. 1 Henan Institute of Science and Technology/Henan Collaborative Innovation Center of Modern Biological Breeding, Xinxiang 453003, Henan, China
    2 Cotton Research Center of Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
  • Received:2019-04-25 Accepted:2019-08-09 Online:2020-01-12 Published:2020-03-04
  • Contact: Hai-Yan HU E-mail:haiyanhuhhy@126.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31601347);Henan Postdoctoral Science Foundation(1902042);Henan Scientific and Technological Research Program(192102110030);Key Research Projects of Henan Higher Education Institutions(19A210013)

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Abstract:

Transcriptional regulation of KUP/HAK/KT potassium transporter gene is an important mechanism of plant response to low potassium stress. Cloning and analysis of promoter of potassium transporter gene in cotton is not only helpful to understand its expression pattern and regulation mechanism, but also important to improve the potassium absorption in cotton. Potassium transporter gene GhHAK5 is a highly expressed in roots and induced by low potassium stress in upland cotton, but the function of its promoter is still unclear. In this study, the 2000 bp promoter fragment of GhHAK5 was cloned from upland cotton variety Baimian 1 by using PCR amplification, and its function was analyzed by GUS histochemical staining and induced expression analysis of GUS under low potassium in pGhHAK5 transgenic Arabidopsis thaliana. In addition to TATA-box, CAAT-box and other basic cis-acting elements, pGhHAK5 also contained a number of cis-acting elements responsive to light, stress, phytohormone and circadian. pGhHAK5 was highly consistent with pGrHAK5 in the number and location of important regulatory elements, and had five root-specific expression regulatory elements (ATAAAAT) and an ARF transcription factor binding site (TGTCNN) involved in transcription regulation under low potassium conditions. GUS histochemical staining of transgenic Arabidopsis thaliana seedlings showed that the leaf veins and vascular tissue of hypocotyl were deeply stained, and the roots were shallowly stained. For mature Arabidopsis thaliana plants, enhanced GUS staining was observed in roots, leaf veins and the vascular tissue of calyx, and weakened GUS staining was observed in stem and pod skin, suggesting that pGhHAK5-driven GUS was mainly expressed in mature roots and vascular tissue of shoots. Induced expression analysis of GUS under low potassium in pGhHAK5 transgenic Arabidopsis thaliana showed that the expression of GUS driven by pGhHAK5 was weak in young roots of Arabidopsis thaliana seedlings, and its expression was not enhanced by low potassium stress. These results suggest that pGhHAK5 might be a potassium-deficient inducible promoter mainly in mature roots. Transcriptome and quantitative real-time PCR analysis showed that GhHAK5 expression in roots was affected by developmental stages, which was consistent with the results of GUS expression driven by pGhHAK5 in Arabidopsis thaliana. These results are helpful to understand the molecular mechanism of GhHAK5 expression regulation, and provide theoretical basis for improving potassium uptake efficiency and breeding potassium efficient varieties in cotton.

Key words: promoter, vascular tissues, potassium transporter, low potassium, upland cotton

Fig. 1

Amplification of GhHAK5 promoter sequence in upland cotton (Gossypium hirsutum L.) 1: PCR products of GhHAK5 promoter; 2: DNA marker (DL2000)."

Table 1

cis-acting elements in promoter of GhHAK5"

元件类型
Element type		 名称
Name		 拷贝数
Copy number		 基序
Motif sequence		 功能
Function
基础元件
Basal
element		 5UTR Py-rich stretch 1 TTTCTTCTCT 高转录水平顺式作用元件
cis-acting element conferring high transcription levels
TATA-box 61 TATA/ATATAT/TTTTA 转录起始位点-30核心启动子元件 Core promoter element around -30 of transcription start
CAAT-box 18 CAATT/CAAT/CCAAT 启动子和增强子区的一般顺式作用元件 Common cis-acting element in promoter and enhancer regions
光
Light		 Box I 1 TTTCAAA 光响应元件 Light responsive element
I-box 1 ATGATATGA 部分光响应元件 Part of a light responsive element
G-Box 1 CACGTT 光响应顺式作用调控元件 cis-acting regulatory element involved in light responsiveness
AT1-motif 1 ATTAATTTTACA 部分光响应模块 Part of a light responsive module
Box II 1 GTGGATATTATAT 部分光响应元件 Part of a light responsive element
生物钟
Circadian		 Circadian 1 CAANNNNATC 昼夜节律顺式作用调控元件 cis-acting regulatory element involved in circadian control
植物激素
Phytohormone		 CGTCA-motif 1 CGTCA 茉莉酸响应顺式作用元件 cis-acting regulatory element involved in the MeJA-responsiveness
TCA-element 1 GAGAAGAATA 水杨酸响应顺式作用元件 cis-acting element
involved in salicylic acid responsiveness
TGA-element 1 AACGAC 生长素响应元件 Auxin-responsive element
ERE 1 ATTTCAAA 乙烯响应元件 Ethylene-responsive element
逆境胁迫
Stress		 Box-W1 1 TTGACC 真菌诱导子响应元件
Fungal elicitor responsive element
HSE 4 AAAAAATTTC 热胁迫响应顺式作用元件 cis-acting element
involved in heat stress responsiveness
其他
Other		 Skn-1_motif 4 GTCAT 胚乳表达相关顺式调控元件 cis-acting regulatory element required for endosperm expression
AT-rich element 1 ATAGAAATCAA AT-rich DNA结合蛋白的结合位点(ATBP-1)
Binding site of AT-rich DNA binding protein
Root-specific motif 5 ATAAAAT 根特异性表达响应元件
Root-specific responsive element

Fig. 2

Sequence analysis of GhHAK5 promoter The nucleotide at position +1 is the ATG start codon, the ATG is indicated in red. Parts of the putative cis-regulatory elements are noted under the sequences in shadow. The one ARF binding site (TGTCNN) is underlined in red, and the five root-specific motifs are bold in red."

Fig. 3

Comparative analysis of the promoter sequence of GhHAK5 and its homologous gene The promoter of potassium transporter gene HAK5 of four species in Gossypium are indicated in bold."

Fig. 4

PCR analysis of T1 generation transgenic Arabidopsis plants M: marker; 1: wild Arabidopsis thaliana (negative control); 2: expression vector pCAMBIA1381Z-pGhHAK5 plasmid (positive control); 3-8: transgenic Arabidopsis thaliana."

Fig. 5

GUS histochemical staining in pGhHAK5 transgenic Arabidopsis thaliana A: wild-type Arabidopsis thaliana seedlings; B: pGhHAK5 transgenic Arabidopsis thaliana; 1-4: Arabidopsis seedlings (1) and enlarged view of its leaf (2), hypocotyl (3) and roots (4); 5-12: the leaf (5), stem (6), flower (7), enlarged view of flower (8), pod (9), enlarged view of pod (10), roots (11), and enlarged view of roots (12) in mature period Arabidopsis thaliana."

Fig. 6

Low potassium stress response analysis in pGhHAK5 transgenic Arabidopsis thaliana WT: wild type Arabidopsis thaliana seedlings; HK: high potassium; LK: low potassium. a: Arabidopsis thaliana seedlings; b: enlarged view of leaf; c: enlarged view of roots."

Fig. 7

Spatio-temporal expression analysis of GhHAK5 in upland cotton (Gossypium hirsutum L.) A: transcriptome analysis of spatio-temporal expression of GhHAK5 in upland cotton; B: quantitative real-time PCR analysis of spatio-temporal expression of GhHAK5 in upland cotton; C: the comparison of roots morphological change at different development stages in cotton; ** Significant at the 0.01 probability level."

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