中华猕猴桃GRAS基因家族鉴定及低温胁迫表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0324

摘要/Abstract

摘要：

GRAS基因家族广泛参与植物生长和逆境响应，而低温是制约猕猴桃生产和分布的重要因素之一，鉴定猕猴桃GRAS基因家族，分析其在低温胁迫中的表达情况，为猕猴桃的抗寒研究和品种选育提供理论依据。以中华猕猴桃‘红阳’基因组为参考进行GRAS家族保守结构域比对，通过对鉴定到的家族成员进行系统进化树、蛋白理化性质、基因结构、蛋白三级结构、蛋白质motif、顺式作用元件、共线性、密码子偏好性和基因表达模式等进行分析。结果表明，猕猴桃基因组共存在79个GRAS家族成员，分属于8个亚家族，且各亚家族基因、蛋白结构有所差异。顺式作用原件分析显示该家族基因参与多种植物激素、生长发育以及胁迫响应。密码子偏好性分析发现，该家族密码子第3位碱基更偏好使用嘧啶类碱基（G/T）。鉴定到6个基因可能参与猕猴桃低温胁迫过程，并进行荧光定量PCR验证了该猜测。该研究补充了猕猴桃GRAS基因家族鉴定分析的空白，为猕猴桃抗寒研究奠定分子基础。

关键词: 猕猴桃, GRAS基因家族, 密码子分析, 低温胁迫

Abstract:

GRAS gene family is widely involved in plant growth and stress response. Low temperature is one of the important factors that restricts the production and distribution of kiwifruit（Actinidia chinensis）. The GRAS gene family of kiwifruit was identified, its expression under low temperature stress was analyzed, which may provide a theoretical basis for the research on cold resistance and variety breeding of kiwifruit. The GRAS gene family conserved domains were compared with the genome of A. chinensis ‘Hongyang’ reference, and the identified family members were analyzed by phylogenetic tree, protein physical, chemical properties, gene structure, protein tertiary structure, protein motif, cis-acting elements, collinearity, codon usage preference and gene expression mode. The results showed that there was a total of 79 GRAS family genes in kiwifruit genome, belonging to 8 subfamilies, and there were differences in gene and protein structures among each subfamily. Analysis of cis-acting elements showed the genes were involved in multiple plant hormone, growth and development, and stress response. Through codon preference analysis, it was found that the third base of GRAS family preferred to use pyrimidine bases G/T. Six genes were identified that may be involved in the low-temperature stress process in kiwifruit, and this hypothesis was validated by fluorescence quantitative PCR.

Key words: kiwifruit, GRAS gene family, codon analysis, low temperature stress

毛可欣, 王海荣, 安淼, 刘腾飞, 王世金, 李健, 李国田. 中华猕猴桃GRAS基因家族鉴定及低温胁迫表达分析[J]. 生物技术通报, 2023, 39(11): 297-307.

MAO Ke-xin, WANG Hai-rong, AN Miao, LIU Teng-fei, WANG Shi-jin, LI Jian, LI Guo-tian. Identification of GRAS Gene Family and Expression Analysis Under Low Temperature Stress in Actinidia chinensis[J]. Biotechnology Bulletin, 2023, 39(11): 297-307.

图/表 12

表1 实时荧光定量PCR引物

Table 1 Primers used for quantitative real-time PCR

基因Gene	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
AcGRAS32	ACATTAGGCTCCCACCACTG	CTCCCGAGTCATTCAACCAT
AcGRAS3	AGTTCCGGATGTTGAGGTTG	TTTGAAGCGGGGTAAGAATG
AcGRAS53	TTCCAATCCTGCAATGAACA	AGGAATGATGCGGATCTGAC
AcGRAS79	GGTCGATGATCTTTCCGTGT	TCCCGAAGAGCTTCAGAAAA
AcGRAS6	GAACGAAGGTTTTCGCTCTG	ACTGGATCCCCTGAGCTTTT
AcGRAS42	AGTCGGCGATGAGCTTAAAA	GGAACCTTGTACAGCCGAAA
Actin	GTGCTCAGTGGTGGTTCAA	GACGCTGTATTTCCTCTCAG

图1 猕猴桃中GRAS基因家族系统发育进化树

Fig. 1 Phylogenetic evolutionary tree of kiwifruit GRAS gene family

图2 GRAS基因家族理化性质

Fig. 2 Physical and chemical properties of GRAS family

图3 GRAS家族的基因结构

Fig. 3 Genetic structure of GRAS family

图4 部分GRAS基因的蛋白三级结构

Fig. 4 Protein tertiary structure of some GRAS genes

图5 GRAS基因家族结构域

Fig. 5 Domain of GRAS family

图6 GRAS基因家族的顺式作用元件

Fig. 6 Cis-acting element of GRAS gene family

图7 GRAS家族共线性分析

Fig. 7 Collinearity analysis of GRAS family

图8 猕猴桃GRAS家族ENC-plot曲线

Fig. 8 ENC-plot curve of kiwifruit GRAS family genes

图9 猕猴桃GRAS家族PR2-plot

Fig. 9 PR2-plot of kiwifruit MYB family genes

图10 GRAS基因在猕猴桃越冬期表达分析 1-3分别表示2020年11月15日、2021年1月15日、2021年3月15日的越冬期猕猴桃枝条

Fig. 10 Analysis of GRAS gene expression in kiwifruit during overwintering 1-3 represents kiwifruit branches in overwintering period on November 15, 2020, January 15, 2021 and March 15, 2021 respectively

图11 低温处理下猕猴桃6个GRAS基因的表达不同字母表示不同处理时间在0.05水平上差异显著。*，**表示处理与对照在0.05和0.01水平差异显著

Fig. 11 Expression analysis of 6 kiwifruit GRAS genes under low temperature treatment Different letters indicate that there are significant differences in genes at the level of 0.05 under different treatment times. *, ** indicated significant difference between treatment and control at 0.05 and 0.01 levels

参考文献 48

[1]	李新伟, 李建强, Soejarto DD. 中国猕猴桃科新异名[J]. 植物分类学报, 2007, 45(5): 633-660.
	Li XW, Li JQ, Soejarto DD. New synonyms in Actinidiaceae from China[J]. Acta Phytotaxon Sin, 2007, 45(5): 633-660.. doi: 10.1360/aps06061 URL
[2]	廖慧苹, 梅兵, 鲍荣粉, 等. 中国猕猴桃资源的利用与品种开发研究[J]. 落叶果树, 2018, 50(3): 33-35.
	Liao HP, Mei B, Bao RF, et al. Study on utilization and variety development of kiwifruit resources in China[J]. Deciduous Fruits, 2018, 50(3): 33-35.
[3]	徐小彪, 张秋明. 中国猕猴桃种质资源的研究与利用[J]. 植物学通报, 2003, 20(6): 648-655.
	Xu XB, Zhang QM. Researches and utilizations of germplasm resource of kiwifruit in China[J]. Chin Bull Bot, 2003, 20(6): 648-655.
[4]	钟彩虹, 黄文俊, 李大卫, 等. 世界猕猴桃产业发展及鲜果贸易动态分析[J]. 中国果树, 2021(7): 101-108.
	Zhong CH, Huang WJ, Li DW, et al. Dynamic analysis of kiwifruit industry development and fresh fruit trade in the world[J]. China Fruits, 2021(7): 101-108.
[5]	屈振江, 周广胜. 中国主栽猕猴桃品种的气候适宜性区划[J]. 中国农业气象, 2017, 38(4): 257-266.
	Qu ZJ, Zhou GS. Regionalization of climatic suitability for major kiwifruit cultivars in China[J]. Chin J Agrometeorology, 2017, 38(4): 257-266.
[6]	黄永红, 史修柱, 李桂云, 等. 2016年泰山南麓猕猴桃冻害调查与分析[J]. 落叶果树, 2016, 48(6): 17-19.
	Huang YH, Shi XZ, Li GY, et al. Investigation and analysis of kiwifruit freezing injury at the southern foot of Mount Tai in 2016[J]. Deciduous Fruits, 2016, 48(6): 17-19.
[7]	李广文, 贺瑶, 李红娟, 等. 陕西宝鸡产区猕猴桃冻害发生规律调查[J]. 西北园艺: 果树, 2018(2): 48-51.
	Li GW, He Y, Li HJ, et al. Investigation on the occurrence law of kiwifruit freezing injury in Baoji production area of Shanxi province[J]. Northwest Hortic, 2018(2): 48-51.
[8]	Aoyanagi T, Ikeya S, Kobayashi A, et al. Gene regulation via the combination of transcription factors in the INDETERMINATE DOMAIN and GRAS families[J]. Genes: Basel, 2020, 11(6): E 613.
[9]	Pysh LD, Wysocka-Diller JW, Camilleri C, et al. The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes[J]. Plant J, 1999, 18(1): 111-119. pmid: 10341448
[10]	Hirano Y, Nakagawa M, Suyama T, et al. Structure of the SHR-SCR heterodimer bound to the BIRD/IDD transcriptional factor JKD[J]. Nat Plants, 2017, 3(3): 1-10.
[11]	Cenci A, Rouard M. Evolutionary analyses of GRAS transcription factors in angiosperms[J]. Front Plant Sci, 2017, 8: 273. doi: 10.3389/fpls.2017.00273 pmid: 28303145
[12]	de Lucas M, Davière JM, Rodríguez-Falcón M, et al. A molecular framework for light and gibberellin control of cell elongation[J]. Nature, 2008, 451(7177): 480-484. doi: 10.1038/nature06520
[13]	Feng SH, Martinez C, Gusmaroli G, et al. Coordinated regulation of Arabidopsis thaliana development by light and gibberellins[J]. Nature, 2008, 451(7177): 475-479. doi: 10.1038/nature06448
[14]	Helariutta Y, Fukaki H, Wysocka-Diller J, et al. The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling[J]. Cell, 2000, 101(5): 555-567. doi: 10.1016/s0092-8674(00)80865-x pmid: 10850497
[15]	Tanabe S, Onodera H, Hara N, et al. The elicitor-responsive gene for a GRAS family protein, CIGR2, suppresses cell death in rice inoculated with rice blast fungus via activation of a heat shock transcription factor, OsHsf23[J]. Biosci Biotechnol Biochem, 2016, 80(1): 145-151. doi: 10.1080/09168451.2015.1075866 URL
[16]	Fode B, Siemsen T, Thurow C, et al. The Arabidopsis GRAS protein SCL14 interacts with class II TGA transcription factors and is essential for the activation of stress-inducible promoters[J]. Plant Cell, 2008, 20(11): 3122-3135. doi: 10.1105/tpc.108.058974 URL
[17]	Achard P, Gong F, Cheminant S, et al. The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism[J]. Plant Cell, 2008, 20(8): 2117-2129. doi: 10.1105/tpc.108.058941 pmid: 18757556
[18]	Achard P, Renou JP, Berthomé R, et al. Plant DELLAs restrain growth and promote survival of adversity by reducing the levels of reactive oxygen species[J]. Curr Biol, 2008, 18(9): 656-660. doi: 10.1016/j.cub.2008.04.034 pmid: 18450450
[19]	牛义岭, 姜秀明, 许向阳. 番茄GRAS转录因子家族基因SIGLD1的克隆及VIGS载体构建[J]. 基因组学与应用生物学, 2016, 35(8): 2155-2160.
	Niu YL, Jiang XM, Xu XY. Cloning and VIGS vector construction of GRAS transcription factors family gene SIGLD1 from tomato[J]. Genom Appl Biol, 2016, 35(8): 2155-2160.
[20]	石瑞, 曹诣斌, 陈文荣, 等. 佛手GRAS基因的克隆及表达分析[J]. 浙江师范大学学报: 自然科学版, 2011, 34(4): 446-451.
	Shi R, Cao YB, Chen WR, et al. On cDNA cloning and expression analysis of GRAS gene in fingered citron[J]. J Zhejiang Norm Univ Nat Sci, 2011, 34(4): 446-451.
[21]	李丽丽, 曾卫军, 李艳红, 等. 独行菜GRAS转录因子家族分析及LaSCL18基因克隆与冷相关性研究[J]. 分子植物育种, 2017, 15(9): 3428-3437.
	Li LL, Zeng WJ, Li YH, et al. Analysis of GRAS family transcription factors, cloning and researching cold tolerance of LaSCL18 gene in Lepidium[J]. Mol Plant Breed, 2017, 15(9): 3428-3437.
[22]	Yang M, Yang Q, Fu T, et al. Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance[J]. Plant Cell Rep, 2011, 30(3): 373-388. doi: 10.1007/s00299-010-0940-7 URL
[23]	周莲洁, 杨中敏, 张富春, 等. 新疆盐穗木GRAS转录因子基因克隆及表达分析[J]. 西北植物学报, 2013, 33(6): 1091-1097.
	Zhou LJ, Yang ZM, Zhang FC, ey al. Expression analysis and cloning of GRAS transcription factor gene from Halostachys caspica[J]. Acta Bot Boreali-Occidentalia Sin, 2013, 33(6): 1091-1097.
[24]	Tian C, Wan P, Sun S, et al. Genome-wide analysis of the GRAS gene family in rice and Arabidopsis[J]. Plant Mol Biol, 2004, 54(4): 519-532. doi: 10.1023/B:PLAN.0000038256.89809.57 URL
[25]	Wang L, Ding X, Gao Y, et al. Genome-wide identification and characterization of GRAS genes in soybean(Glycine max)[J]. BMC Plant Biol, 2020, 20(1): 415. doi: 10.1186/s12870-020-02636-5
[26]	To VT, Shi Q, Zhang Y, et al. Genome-wide analysis of the GRAS gene family in barley(Hordeum vulgare L.)[J]. Genes: Basel, 2020, 11(5): E553.
[27]	唐瑞, 韩妮, 虎亚静, 等. 黄瓜GRAS家族全基因组鉴定与表达分析[J]. 分子植物育种, 2020, 19(13): 4242-4251.
	Tang R, Han N, Hu YJ, et al. Genome-wide identification and expression analysis of GRAS genes in cucumber[J]. Mol Plant Breed, 2020, 19(13): 4242-4251.
[28]	张文慧, 何光鑫, 王子鑫, 等. 绿豆GRAS基因家族鉴定及其非生物胁迫下的表达模式分析[J]. 农业生物技术学报, 2022, 30(5): 861-872.
	Zhang WH, He GX, Wang ZX, et al. Identification of GRAS gene family and its expression pattern analysis under abiotic stress in Vigna radiata[J]. J Agric Biotechnol, 2022, 30(5): 861-872.
[29]	刘凯, 赵星哲, 王红霞, 等. 核桃JrGRAS基因家族鉴定与响应低温胁迫基因筛选[J]. 河北农业大学学报, 2021, 44(2): 27-40.
	Liu K, Zhao XZ, Wang HX, et al. Identification of walnut JrGRAS gene family and screening of genes in response to low temperature stress[J]. J Agric Univ Hebei, 2021, 44(2): 27-40.
[30]	李晨, 赵雪惠, 王庆杰, 等. 桃GRAS家族全基因组鉴定与响应UV-B的表达模式分析[J]. 中国农业科学, 2019, 52(24): 4567-4581. doi: 10.3864/j.issn.0578-1752.2019.24.011
	Li C, Zhao XH, Wang QJ, et al. Genome identification of PpGRAS family and expression pattern analysis of responding to UV-B in peach[J]. Sci Agric Sin, 2019, 52(24): 4567-4581.
[31]	Yue J, Liu J, Tang W, et al. Kiwifruit genome database(KGD): a comprehensive resource for kiwifruit genomics[J]. Hortic Res, 2020, 7: 117. doi: 10.1038/s41438-020-0338-9
[32]	Camacho C, Coulouris G, Avagyan V, et al. BLAST+: architecture and applications[J]. BMC Bioinformatics, 2009, 10: 421. doi: 10.1186/1471-2105-10-421 pmid: 20003500
[33]	Tian F, Yang DC, Meng YQ, et al. PlantRegMap: charting functional regulatory maps in plants[J]. Nucleic Acids Res, 2020, 48(D1): D1104-D1113.
[34]	Lu S, Wang J, Chitsaz F, et al. CDD/SPARCLE: the conserved domain database in 2020[J]. Nucleic Acids Res, 2020, 48(d1): D265-D268. doi: 10.1093/nar/gkz991 URL
[35]	Thompson JD, Gibson TJ, Higgins DG. Multiple sequence alignment using ClustalW and ClustalX[J]. Curr Protoc Bioinformatics, 2002, Chapter 2: Unit 2.3.
[36]	Chen C, Chen H, Zhang Y, et al. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Mol Plant, 2020, 13(8): 1194-1202. doi: S1674-2052(20)30187-8 pmid: 32585190
[37]	Wang YP, Tang HB, Debarry JD, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity[J]. Nucleic Acids Res, 2012, 40(7): e49. doi: 10.1093/nar/gkr1293 URL
[38]	Novembre JA. Accounting for background nucleotide composition when measuring codon usage bias[J]. Mol Biol Evol, 2002, 19(8): 1390-1394. pmid: 12140252
[39]	Sueoka N. Near homogeneity of PR2-bias fingerprints in the human genome and their implications in phylogenetic analyses[J]. J Mol Evol, 2001, 53(4): 469-476. doi: 10.1007/s002390010237 URL
[40]	Hakoshima T. Structural basis of the specific interactions of GRAS family proteins[J]. FEBS Lett, 2018, 592(4): 489-501. doi: 10.1002/1873-3468.12987 pmid: 29364510
[41]	Bolle C. The role of GRAS proteins in plant signal transduction and development[J]. Planta, 2004, 218(5): 683-692. doi: 10.1007/s00425-004-1203-z pmid: 14760535
[42]	Sun TP. The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants[J]. Curr Biol, 2011, 21(9): 338-345. doi: 10.1016/j.cub.2011.01.036 URL
[43]	Wang LJ, Roossinck MJ. Comparative analysis of expressed sequences reveals a conserved pattern of optimal codon usage in plants[J]. Plant Mol Biol, 2006, 61(4): 699-710. doi: 10.1007/s11103-006-0041-8 URL
[44]	Zhang H, Li J, Wang RX, et al. Comparative analysis of expansin gene codon usage patterns among eight plant species[J]. J Biomol Struct Dyn, 2019, 37(4): 910-917. doi: 10.1080/07391102.2018.1442746 pmid: 29480091
[45]	侯倩倩. 外源基因在产甘油假丝酵母中的表达策略及应用研究[D]. 无锡: 江南大学, 2019.
	Hou QQ. Expression strategy and application of exogenous genes in Candida glycerinogenes[D]. Wuxi: Jiangnan University, 2019.
[46]	Yan XW, Hoek TA, Vale RD, et al. Dynamics of translation of single mRNA molecules in vivo[J]. Cell, 2016, 165(4): 976-989. doi: 10.1016/j.cell.2016.04.034 URL
[47]	殷龙飞, 张中保, 于荣, 等. 植物GRAS家族蛋白结构和功能的研究进展[J]. 分子植物育种, 2019, 17(19): 6323-6331.
	Yin LF, Zhang ZB, Yu R, et al. Progress of the structural and functional analysis of GRAS gene in plants[J]. Mol Plant Breed, 2019, 17(19): 6323-6331.
[48]	Wang Z, Wong DCJ, Wang Y, et al. GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response[J]. Plant Physiol, 2021, 186(3): 1660-1678. doi: 10.1093/plphys/kiab142 pmid: 33752238