Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (19): 3404-3416.doi: 10.3864/j.issn.0578-1752.2019.19.011

• HORTICULTURE • Previous Articles     Next Articles

Cloning, Subcellular Localization and Expression Analysis of CitPG34 in Citrus

GE Ting,HUANG Xue,XIE RangJin()   

  1. Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712
  • Received:2019-05-05 Accepted:2019-07-12 Online:2019-10-01 Published:2019-10-11
  • Contact: RangJin XIE E-mail:xierangjin@cric.cn

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Abstract:

【Objective】 Polygalacturonases (PGs) play important roles in plant growth and development as well as organ abscission by degrading pectin in cell wall. In this study, a citrus PG gene (i.e., CitPG34) and its promoter (CitPG34-P) were cloned and expression analyzed based on our previous data, which would provide a basis for further elucidating the function of CitPG34 in citrus fruitlet abscission.【Method】 The full length of CitPG34 gene and its promoter was cloned from ‘Tarcocco’ blood orange (Citrus sinensis L. Osbeck). The protein characteristics and cis-acting elements on promoter were analyzed by ProtParam, Cello, CLUSTALX, MEGA5.2, and PlantCARE, etc. The gene expression level was detected by real-time Quantitative PCR (qRT-PCR). The PCAMBIA1302-CitPG34-GFP fusion protein expression vector for subcellular localization and CitPG34-P expression vector (CitPG34-P::gus) for promoter activity analysis were constructed by homologous recombination, respectively.【Result】 The ORF of CitPG34 was 1 194 bp in length, encoding 397 amino acids. The predicted molecular weight of CitPG34 was 41.47 kD, the theoretical pI was 5.19, and the instability coefficient was 30.23, indicating that CitPG34 belonged to stable protein. TMHMM analysis showed that CitPG34 was a transmembrane protein, the transmembrane domain locating between the amino acid residue 7 and 29. In the secondary structure of CitPG34, the alpha-helix structure, extended chain and random coil account for 15.37%, 29.72% and 54.91%, respectively, which were nearly consistent with its tertiary structure. NJ tree analysis showed that CitPG34 was close to PcPG3 (BAF42034), a pear PG, indicating that it might be related to fruit abscission and softening. qPCR analysis showed that CitPG34 dominantly expressed in flowers, followed by roots, leaves, abscission zone A (AZ A) and C (AZ C), and almost undetected in fruits. In AZ A, the expression level of CitPG34 was significantly up-regulated by ACC, whereas inhibited by IAA, showing the role in citrus fruitlet abscission. Subcellular localization revealed that CitPG34 was mainly located in cell wall. A 2 075 bp promoter sequence of CitPG34 was cloned, which contained several cis-regulatory elements, including TATA-box, enhancer CAAT-box and ABRE, etc. GUS histochemical staining revealed that the GUS activity in vein and trichomes was remarkably up-regulated by ethylene. 【Conclusion】 The ORF length of CitPG34 gene was 1 194 bp, encoding 397 amino acids. CitPG34 was mainly located in cell wall. qPCR analysis showed that CitPG34 dominantly expressed in flowers and was significantly associated with citrus fruitlet abscission. Taken together, these results indicated that CitPG34 played important roles in citrus fruitlet abscission and flower development.

Key words: citrus, Polygalacturonases, gene expression, subcellular localization, promoter, fruitlet abscission

Fig. 1

Tissues used for the tissue specific expression of CitPG34 A: Root (Rootstock); B: Leaves; C: Fruitlet"

Table 1

Primers used in this study"

引物名称 Primers name 引物序列 Sequence of primers (5′→3′) 目的 Purpose
OE-CitPG34-F GCTTTGAAACCATGTCAACATC 基因全长克隆
Full-length gene cloning
OE-CitPG34-R TAGGAACTGCCTCTAAGTAAGC
CitPG34-F ATTAGCATCCGTAGATAGCC 实时荧光定量
Real-time PCR
CitPG34-R CACAGTCATCACCAGTCCC
P- CitPG34-F TGATTACGCCAAGCTTACCGTCTATGCTACTGATGTGA 启动子克隆
Promoter cloning
P- CitPG34-R AGGGACTGACCACCCTGATGTTGACATGGTTTCAAAGC
Cit-Actin-F CCCCATCGTTACCGTCCAG 内参基因
Actin gene
Cit-Actin-R CGCCTTGCCAGTTGAATATCC
Sub-CitPG34-F GGGGGACTCTTGACCATGTCAACATCAATGTCTTTGCC 亚细胞定位
Subcellular localization
sub-CitPG34-R CTAGTCAGATCTACCATGACACCAGAACCACCACCAGA
ACCAGTCTTCAAGCAACTATTTGGAAG
pCAMBIA1302-F CCTTCACCCTCTCCACTGACAG 亚细胞定位
Subcellular localization
pCAMBIA1302-R CCTTCGCAAGACCCTTCCTCTA

Fig. 2

PCR amplified product M: DL2000 maker. A: PCR amplified product of CitPG34; B: PCR amplified product of CitPG34 promoter"

Fig. 3

Amino acid sequence alignment of CitPG34 with other PG proteins I: SPNTDG domain, II: GDDC domain, III: CGPGHG domain, IV: RIK domain"

Fig. 4

Phylogenetic tree of plant PGs"

Fig. 5

Expression levels of CitPG34 in different tissues Different lowercase letters indicate significant difference (P<0.05). The same as below"

Fig. 6

Expression levels of CitPG34 during citrus fruitlet abscission"

Fig. 7

The subcellular localization of CitPG34 protein A, B, C, D: Epidermal cells were not plasmolyzed; E, F, G, H: Epidermal cells were not plasmolyzed; Scale = 100 μm"

Table 2

The major cis-acting elements prediction of CitPG34 gene promoter"

顺式作用元件
cis-element		 序列
Sequence		 功能
Function		 数量
Number
ABRE CACGTG; ACGTG; AACCCGG 脱落酸应答相关的顺式作用元件
Cis-acting element involved in the abscisic acid responsiveness		 7
ARE AAACCA 厌氧诱导必须顺式作用元件
Cis-acting regulatory element essential for the anaerobic induction		 2
CGTCA-motif CGTCA 甲基茉莉酸应答相关的顺式作用元件
Cis-acting regulatory element involved in the MeJA-responsiveness		 2
MBS CAACTG 参与干旱诱导的MYB结合位点
MYB binding site involved in drought-inducibility		 3
MBSI aaaAaaC(G/C)GTTA 类黄酮生物合成基因调控相关的MYB结合位点
MYB binding site involved in flavonoid biosynthetic genes regulation		 1
MYB TAACCA 赤霉素作用元件 Gibberellin action element 3
MYB-like sequence TAACCA 赤霉素作用元件 Gibberellin action element 3
MYC CATTTG; CATGTG MYC结合位点 MYC binding site 2
TC-rich repeats ATTCTCTAAC 胁迫响应元件
Cis-acting element involved in defense and stress responsiveness		 1
TCA-element CCATCTTTTT 水杨酸应答元件
Cis-acting element involved in salicylic acid responsiveness		 1
TGACG-motif TGACG 茉莉酸甲酯调控元件
Cis-acting regulatory element involved in the MeJA-responsiveness		 2
WUN-motif AAATTTCCT 机械伤应答元件 wound-responsive element 1
as-1 TGACG 茉莉酸甲酯调控元件
Cis-acting regulatory element involved in the MeJA-responsiveness		 2
Box-W1 TTGACC 真菌诱导应答元件 Fungal induced response elements 1

Fig. 8

Histochemical staining of GUS expression in CitPG34 promoters"

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