Cloning and Sequence Analysis of 5'UTR Intron of  GhFAD2-1 Gene from Cotton (Gossypium hirsutum L.)

doi:10.6048/j.issn.1001-4330.2018.01.001

Abstract

Abstract: 【Objective】 Cloning and sequence analysis of the 5'UTR intron of the delta 12- oleic acid desaturase gene GhFAD2-1 would lay the foundation for the study of the expression and regulation of GhFAD2-1 . 【Method】 In this study, the 5'UTR sequence of GhFAD2-1 was cloned by 5'RACE technology. And then, 5'UTR intron of GhFAD2-1 was cloned in combination with the cotton genome sequence. Cis elements were also analyzed by PLACE and other bioinformatics software.【Result】 The GhFAD2-1 5'UTR sequence 77 bp was obtained from Gossypium hirsutum using 5'RACE technique. The result showed that the transcription start site is T based on a combination of 5'UTR and genome sequence analysis. GhFAD2-1 5'UTR contained a full-length 1,103 bp intron sequence in the A genome of cotton. The full-length sequence of GhFAD2-1 5'UTR intron was 1,111 bp in D genome. The cleavage sites of 5' UTR intron were AA-GG, CA-GC, respectively. Cis element analysis showed that the intron included some typical function and optical response related elements and elements related to hormone regulation or stress response.【Conclusion】 5'UTR intron sequences of GhFAD2-1 gene were cloned from the A and D genome, respectively. The transcription start site and the cleavage sites of 5' UTR intron were also identified. The results have laid a foundation to further study expression and regulation of GhFAD2-1 at the molecular level.

Key words: cotton; GhFAD2-1 gene; 5' UTR intron; cloning; sequence analysis

摘要： 【目的】 对棉花^Δ12-油酸去饱和酶基因GhFAD2-1 5'UTR区的内含子进行克隆和序列分析,为研究GhFAD2-1的表达调控奠定基础。【方法】 利用5'RACE 技术,克隆GhFAD2-1 的5'UTR序列,结合棉花基因组序列,克隆GhFAD2-1 5'UTR内含子,并利用PLACE等生物信息学软件对其顺式作用原件进行分析。【结果】 棉花A、D基因组的GhFAD2-1 5' UTR中各含一内含子序列,全长分别为1 103 bp、1 111 bp;GhFAD2-1成熟mRNA的5' UTR为77bp,转录的起点碱基为T;5' UTR内含子两个剪切位点分别AA-GG、CA-GC。该内含子包括一些典型的与光响应相关的作用元件,以及与激素和胁迫因素相关的应答元件等。【结论】 克隆获得了棉花A、D基因组的GhFAD2-1基因5'UTR内含子序列;明确其转录的起点碱基及5' UTR内含子的剪切位点,为进一步在分子水平上研究GhFAD2-1功能及其表达调控规律,为植物的遗传改良奠定了基础。

关键词: 棉花, GhFAD2-1基因, 5&apos, UTR内含子, 克隆, 序列分析

CLC Number:

S562
S188

SUN Liang, WEN Feng, LIU Feng, ZHANG Xin-yu, SUN Jie. Cloning and Sequence Analysis of 5'UTR Intron of GhFAD2-1 Gene from Cotton (Gossypium hirsutum L.)[J]. Xinjiang Agricultural Sciences, 2018, 55(1): 1-8.

孙亮,文凤,刘峰,张新宇,孙杰. 棉花GhFAD2-1基因5’UTR内含子的克隆与序列分析[J]. 新疆农业科学, 2018, 55(1): 1-8.

References 16

[1]	Shanklin, J., & Cahoon, E. B. (1998). Desaturation and related modifications of fatty acids1. Annual Review of Plant Physiology & Plant Molecular Biology , 49(49): 611-641.
[2]	Liu, Q., Brubaker, C. L., Green, A. G., Marshall, D. R., Sharp, P. J., & Singh, S. P. (2001). Evolution of the fad2-1 fatty acid desaturase 5' utr intron and the molecular systematics of gossypium (malvaceae). American Journal of Botany , 88(1): 92-102.
[3]	牛俊奇,吴朝兴,杨丽涛,等. 甘蔗SoNIN1基因结构及其内含子信息分析[J]. 生物技术通报,2014,(12):147-152.Niu Jun-qi, Wu Chao-xing, Yang Li-tao, et al. (2014). Analysis of Genomic Structure and Introns of SoNIN1 Gene from Sugarcane [J]. Biotechnology Bulletin , (12):147-152. (in Chinese)
[4]	李秀兰,姜曰水. 高等植物细胞器Ⅱ类内含子剪接的研究进展[J]. 植物生理学报,2017,53(8):1 365-1 371.LI Xiu-Lan, JIANG Yue-Shui. (2017). Research progress of group II intron splicing in higher plant organelles [J]. Plant Physiology Journal , 53(8):1,365-1,371. (in Chinese)
[5]	Zhang, J., Liu, H., Sun, J., Li, B., Zhu, Q., & Chen, S., et al. (2012). Arabidopsis fatty acid desaturase fad2 is required for salt tolerance during seed germination and early seedling growth. Plos One , 7(1): e30355.
[6]	Matteucci, M., D'Angeli, S., Errico, S., Lamanna, R., Perrotta, G., & Altamura, M. M. (2011). Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of olea europaea l. genotypes with different cold hardiness. Journal of Experimental Botany , 62(10): 3,403-3,402.
[7]	Kargiotidou, A., Deli, D., Galanopoulou, D., Tsaftaris, A., & Farmaki, T. (2008). Low temperature and light regulate delta 12 fatty acid desaturases (fad2) at a transcriptional level in cotton (gossypium hirsutum). Journal of Experimental Botany , 59(8): 2,043-2,056.
[8]	Donath, M., Mendel, R., Cerff, R., & Martin, W. (1995). Intron-dependent transient expression of the maize gapa1, gene. Plant Molecular Biology ,28(4): 667-676.
[9]	Callis, J., Fromm, M., & Walbot, V. (1987). Introns increase gene expression in cultured maize cells. Genes & Development , 1(10): 1,183-1,200.
[10]	Maas, C., Laufs, J., Grant, S., Korfhage, C., & Werr, W. (1991). The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1,000-fold. Plant Molecular Biology , 16(2): 199-207.
[11]	Morello, L., Bardini, M., Cricrì, M., Sala, F., & Breviario, D. (2006). Functional analysis of dna sequences controlling the expression of the rice oscdpk2, gene. Planta , 223(3): 479-491.
[12]	Jeon JongSeong, Lee SiChul, Jung KiHong, Jun SungHoon, Kim ChanHong, & An GynHeung. (2000). Tissue-preferential expression of a rice α-tubulin gene, ostuba1, mediated by the first intron. Plant Physiology , 123(3): 1,005-1,014.
[13]	Dean, C., Favreau, M., Bond-Nutter, D., Bedbrook, J., & Dunsmuir, P. (1989). Sequences downstream of translation start regulate quantitative expression of two petunia rbcs genes. Plant Cell , 1(2): 201-208.
[14]	Leon, P., & Walbot, V. (1991). Transient gene expression in protoplasts of phaseolus vulgaris isolated from a cell suspension culture. Plant Physiology , 95(3): 968-972.
[15]	Bourdon, V., Harvey, A., & Lonsdale, D. M. (2001). Introns and their positions affect the translational activity of mrna in plant cells. Embo Reports , 2(5): 394-398.
[16]	Vasil, V., Clancy, M., Ferl, R. J., Vasil, I. K., & Hannah, L. C. (1989). Increased gene expression by the first intron of maize shrunken-1 locus in grass species. Plant Physiology , 91(4): 1,575-1,579.