Cloning, Physical Location and Expression Analysis of HbGRF in Hevea brasiliensis

doi:10.3969/j.issn.1000-2561.2020.09.001

Abstract

Abstract:

Growth-regulating factor (GRF) is a plant-specific transcription factor. The transcription factor family has a large number of members and plays an important role in plant growth and development. In this study, the excellent clone ‘Reyan 7-33-97’ was used as the experimental material, and the HbGRF genes were cloned by RT-PCR. The bioinformatics method was used to study its gene length, physicochemical properties, basic structure and evolutionary relationship. The physical localization analysis was carried out by in situ PCR and fluorescence in situ hybridization. The expression pattern of HbGRF genes in rubber trees was analyzed by qRT-PCR. Three GRF genes were cloned into the female flowers of rubber trees, named HbGRF1, HbGRF2 and HbGRF3, respectively. The protein length was 609 aa, 384 aa and 494 aa with molecular weight of 65.663 kDa, 41.188 kDa and 52.858 kDa, respectively, and they were all unstable protein. All the three members had the complete characteristic domains WRC and QLQ of GRF gene family. Evolutionary analysis showed that the three genes belonged to three different groups. HbGRF1, HbGRF2 and HbGRF3 were located on the long arm of chromosome 11, the short arm of chromosome 5 and the long arm of chromosome 9, and the average percentage distance between the signal site and the centromere was 77.65, 42.66 and 65.27, respectively. The expression results showed that the three HbGRF genes in rubber trees were highly expressed in the vigorous growth tissues like stem tips and female flowers. Under the treatment of exogenous GA and ABA, the expression levels of the three genes increased first, and the expression levels at 48 h were basically the same as those under the initial conditions. The three genes had obvious expression specificity in stem tips and female flowers, which may play important roles in flower bud differentiation and female flower development. This study would provide a theoretical basis and materials for further exploration of the function of HbGRF, and provide molecular cytology basis for accurate breeding of rubber trees.

Key words: Hevea brasiliensis, GRF gene family, gene mapping, expression analysis

CLC Number:

S794.1

ZHANG Yuhang,PAN Ranran,LI Fei,TAO Zhiqiang,WANG Ying,GAO Heqiong,ZHUANG Nansheng. Cloning, Physical Location and Expression Analysis of HbGRF in Hevea brasiliensis[J]. Chinese Journal of Tropical Crops, 2020, 41(9): 1723-1732.

Figures/Tables 12

Tab. 1 Primers sequences for PCR

基因 Gene	上游引物(5ʹ-3ʹ) Forward primer (5ʹ-3ʹ)	下游引物(5ʹ-3ʹ) Reverse primer (5ʹ-3ʹ)	用途 Use
HbGRF1	AAATAGTTACAAAGCAGGC	TTCTTCAGTGTAGAGGTGGTG	基因克隆
	GCTGCCTCTTCCTCATC	AACACCTTGGACTACGA	基因定位
	AGAAAGCCTGTGGAAGGTCA	CCACCACAGCTGCTGAAATA	定量分析
HbGRF2	AAGTTGCTGGTAAGAACAAG	AAAGATGCCAAAACTCCTCCT	基因克隆
	GCACCCTTTATGTTTCC	CAGCAAGATTTGTTAGTAGAG	基因定位
	CAGGTACATGTTGGCTGGTG	ATACGGTGGAGAGCGAAGAA	定量分析
HbGRF3	TCACTACCGTCCGTTTCCATC	CAACCACGGCAATCCAATC	基因克隆
	ACCGCATAATAACCAAT	GTTCAAGAAAGCCTGTG	基因定位
	AGATCCAAATGGGGTTAGGC	GTGTAGCCCCTGTCCAAGAA	定量分析
18S rRNA	GCTCGAAGACGATCAGATACC	TTCAGCCTTGCGACCATAC	定量分析

Tab. 2 Analysis of composition and physicochemical properties of HbGRF gene proteins sequence

名称 Name	分子式 Molecular formula	氨基酸Amino acid	分子量 Molecular weight/kDa	等电点 pI	蛋白质不稳定指数 Protein instability index		脂肪系数 Fat coefficient	疏水性 Hydrophobicity	亚细胞定位 Subcellular localization	起始密码子 Start codon	终止密码子 Stop codon
名称 Name	分子式 Molecular formula	氨基酸Amino acid	分子量 Molecular weight/kDa	等电点 pI	数值 Value	评价 Evaluate	脂肪系数 Fat coefficient	疏水性 Hydrophobicity	亚细胞定位 Subcellular localization	起始密码子 Start codon	终止密码子 Stop codon
GRF1	C₂₂₇₉H₃₅₄₀N₆₆₈O₇₄₅S₂₀	609	65.663	8.48	51.08	不稳定	69.03	-0.541	细胞核	ATG	TAA
GRF2	C₁₇₉₈H₂₈₀₆N₅₂₄O₅₆₅S₁₂	384	41.188	8.77	56.06	不稳定	62.03	-0.580	细胞核	ATG	TGA
GRF3	C₂₂₇₉H₃₅₄₀N₆₆₈O₇₄₅S₂₀	494	52.858	6.09	51.27	不稳定	57.98	-0.582	细胞核	ATG	TAG

Fig. 1 Prediction of secondary structure of HbGRF proteins in H. brasiliensis Red stripe: spiral structure; Blue stripe: chain; Yellow stripe: hidden structure; Green stripe: irregular structure; Red small square: protein binding area; Yellow small circle: DNA binding area; Purple small circle: RNA binding area; Gray small circle: nucleotide binding area.

Fig. 2 Multiple alignment of acid sequences of HbGRF with other species

Fig. 3 Phylogenetic tree Analysis of HbGRF proteins in H. brasiliensis and other GRF proteins

Fig. 4 IS-PCR with HbGRF1 at chromosomes on different phase of mitosis of ‘Reyan 7-33-97’ A: Interphase cell; B: Prometaphase cell; C: Metaphase cell; D: Negative control; E-F: Metaphase cell division and its karyotype; Arrows indicated the signal of HbGRF1.

Fig. 5 IS-PCR with HbGRF2 at chromosomes on different phase of mitosis of ‘Reyan 7-33-97’ A: Interphase cell; B: Prometaphase cell; C: Metaphase cell; D: Negative control; E-F: Metaphase cell division and its karyotype; Arrows indicated the signal of HbGRF2.

Fig. 6 FISH with HbGRF3 at chromosomes on different phase of mitosis of ‘Reyan 7-33-97’ A: Interphase cell; B: Prometaphase cell; C: Metaphase cell; D: Negative control; E-F: Metaphase cell division and its karyotype; Arrows indicated the signal of HbGRF3.

Fig. 7 Idiogram showing the location of three HbGRF genes on genotype ‘Reyan 7-33-97’

Fig. 8 Expression of HbGRF genes in different tissues of H. brasiliensis

Fig. 9 Expression analysis of HbGRF genes of H. brasiliensis in different periods of inflorescence development

Fig. 10 Effect of exogenous hormone treatment on HbGRF expression A-C: GA treatment; D-F: ABA treatment.

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