海岛棉CAD和CAD-Like基因家族的鉴定、表达及其对大丽轮枝菌的响应

doi:10.3864/j.issn.0578-1752.2023.19.005

摘要/Abstract

摘要：

【目的】肉桂醇脱氢酶（CAD）是木质素合成途径中的关键酶，在增强植物机械强度、抵御病原菌入侵等方面发挥重要作用。鉴定海岛棉GbCAD、GbCAD-LIKE（GbCADL）基因家族成员，并对其表达特征及其在黄萎病抗性中的作用进行分析，为棉花抗黄萎病机制的解析及抗病育种提供参考。【方法】利用生物信息学方法对海岛棉GbCAD、GbCADL基因家族成员进行鉴定，并对其染色体定位、系统发育关系、基因结构、启动子顺式元件的预测进行系统分析。通过获得公开释放的转录组数据和实时荧光定量聚合酶链式反应（qRT-PCR）分析GbCAD基因及GbCADL基因的表达特征。利用病毒诱导的基因沉默（VIGS）技术对GbCAD基因及GbCADL基因进行功能分析。【结果】从海岛棉中鉴定到25个GbCAD基因（分布在10条染色体）和34个GbCADL基因（分布在17条染色体）。GbCAD基因分为3个亚组，GbCADL基因分为4个亚组，同一个组内基因含有相似的外显子-内含子结构和保守结构域。GbCAD基因和GbCADL基因具有不同的转录表达特征，其启动子中含有多种激素和胁迫响应元件。转录组数据和qRT-PCR显示，GbCAD10A/D、GbCADL4A/D、GbCADL5A/D、GbCADL6A/D、GbCADL7A/D的表达受大丽轮枝菌诱导，尤其是GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D的表达在大丽轮枝菌处理后显著增加。利用VIGS技术，将显著受大丽轮枝菌诱导表达的GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D分别在棉花中沉默，分析VIGS株系对大丽轮枝菌抗性的变化。结果显示，与对照植株相比，VIGS株系对大丽轮枝菌的抗性显著降低。二氨基联苯胺（DAB）染色结果显示，在未受到黄萎病侵染时，VIGS植株和对照植株的染色程度无显著差异；在大丽轮枝菌处理6 h后，与对照植株相比，沉默株系GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D均表现出较深的着色，表明在沉默株系中有较高的活性氧累积。茎的切片结果显示，大丽轮枝菌处理后，TRV:GbCAD10A/D、TRV:GbCADL4A/D、TRV:GbCADL6A/D、TRV:GbCADL7A/D沉默株系的茎维管组织表现出明显的深褐色，表明其对大丽轮枝菌的抗性显著降低。【结论】抑制GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D的表达可以显著降低棉花对大丽轮枝菌的抗性。

关键词: 海岛棉, 肉桂醇脱氢酶, 基因家族, 表达特征, 基因沉默, 大丽轮枝菌

Abstract:

【Objective】Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin synthesis pathway, which plays an important role in enhancing plant mechanical strength and resisting pathogen invasion. The aim of this study is to identify CAD and CAD-Like (CADL) gene family members in Gossypium barbadense and to analyze their expression characteristics and their role in Verticillium wilt resistance, which provides reference for the mechanism elucidation and disease resistance breeding of cotton against Verticillium wilt. 【Method】The CAD and CADL gene family members in G. barbadense genome were identified by bioinformatics method, and their chromosomal location, phylogenetic relationship, gene structure and promoter cis-element prediction were systematically analyzed. The expression characteristics of GbCAD and GbCADL were analyzed by obtaining publicly released transcriptome data and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Functional analysis of GbCAD and GbCADL genes was performed by viral-induced gene silencing (VIGS) technique. 【Result】A total of 25 GbCAD and 34 GbCADL genes were identified from G. barbadense and distributed on 10 and 17 different chromosomes, respectively. GbCAD and GbCADL genes are divided into 3 and 4 subgroups, respectively. The genes in the same group contain similar exon-intron structures and conserved domains. GbCAD and GbCADL genes have different transcriptional expression characteristics, and the promoters of GbCAD and GbCADL genes contain various hormone response elements and stress response elements. Transcriptome data and qRT-PCR showed that the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL5A/D, GbCADL6A/D, and GbCADL7A/D were induced by Verticillium dahliae, especially the GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D indicated significant increased expressions under V. dahliae treatment. The genes of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D were respectively silenced in cotton by virus-induced gene silencing (VIGS) technology, to analyze the changes of VIGS plant lines against V. dahliae treatment. The results showed that, compared with the control plants, the VIGS plant lines indicated significant decreased resistance to V. dahliae. The results of diaminobenzidine (DAB) histochemical stain displayed that, both control and VIGS plants showed similar normal phenotype without V. dahliae addition; after 6 h treatment of V. dahliae, the VIGS plant lines silencing GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D expressions demonstrated a deeper brown coloring, indicating a higher reactive oxygen species (ROS) accumulation in the VIGS plant lines. The results of stem sectioning showed that, the stem vascular tissues of VIGS plant lines TRV:GbCAD10A/D, TRV:GbCADL4A/D, TRV:GbCADL6A/D, and TRV:GbCADL7A/D showed obvious dark brown enrichment after V. dahliae treatment, indicating the significant decreased resistance to V. dahliae. 【Conclusion】 Suppressing the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D could significantly reduce the cotton resistance to V. dahliae.

Key words: Gossypium barbadense, cinnamyl alcohol dehydrogenase, gene family, expression characteristics, gene silencing, Verticillium dahliae

张钰佳, 崔凯文, 段力升, 曹爱萍, 谢全亮, 沈海涛, 王斐, 李鸿彬. 海岛棉CAD和CAD-Like基因家族的鉴定、表达及其对大丽轮枝菌的响应[J]. 中国农业科学, 2023, 56(19): 3759-3771.

ZHANG YuJia, CUI KaiWen, DUAN LiSheng, CAO AiPing, XIE QuanLiang, SHEN HaiTao, WANG Fei, LI HongBin. Identification and Expression of CAD and CAD-Like Gene Families from Gossypium barbadense and Their Response to Verticillium dahliae[J]. Scientia Agricultura Sinica, 2023, 56(19): 3759-3771.

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参考文献 44

[1]	SONG R R, LI J P, XIE C J, YANG X Y, JIAN W, YANG X Y. An overview of the molecular genetics of plant resistance to the Verticillium wilt pathogen Verticillium dahliae. International Journal of Molecular Sciences, 2020, 21(3): 1120. doi: 10.3390/ijms21031120
[2]	马峙英, 李兴红, 孙济中, 刘金兰. 棉花黄萎病菌致病力分化与寄主抗病性遗传研究进展. 棉花学报, 1996, 8(4): 172-176.
	MA Z Y, LI X H, SUN J Z, LIU J L. Review on the differentiation of V. alboatrum and V. dahliae and the resistance inheritance in cotton. Acta Gossypii Sinica, 1996, 8(4): 172-176. (in Chinese)
[3]	NOVAES E, KIRST M, CHIANG V, WINTER-SEDEROFF H, SEDEROFF R. Lignin and biomass: A negative correlation for wood formation and lignin content in trees. Plant Physiology, 2010, 154(2): 555-561. doi: 10.1104/pp.110.161281 pmid: 20921184
[4]	RENCORET J, GUTIÉRREZ A, NIETO L, JIMÉNEZ-BARBERO J, FAULDS C B, KIM H, RALPH J, MARTÍNEZ Á T, DEL RÍO J C. Lignin composition and structure in young versus adult Eucalyptus globulus plants. Plant Physiology, 2011, 155(2): 667-682. doi: 10.1104/pp.110.167254
[5]	M C FADDEN H G, CHAPPLE R, FEYTER R D, DENNIS E. Expression of pathogenesis-related genes in cotton stems in response to infection by Verticillium dahliae. Physiological and Molecular Plant Pathology, 2001, 58(3): 119-131. doi: 10.1006/pmpp.2001.0320
[6]	MOTTIAR Y, VANHOLME R, BOERJAN W, RALPH J, MANSFIELD S D. Designer lignins: Harnessing the plasticity of lignification. Current Opinion in Biotechnology, 2016, 37: 190-200. doi: S0958-1669(15)00162-7 pmid: 26775114
[7]	DONG N Q, LIN H X. Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions. Journal of Integrative Plant Biology, 2021, 63(1): 180-209. doi: 10.1111/jipb.v63.1
[8]	耿飒, 徐存拴, 李玉昌. 木质素的生物合成及其调控研究进展. 西北植物学报, 2003, 23(1): 171-181.
	GENG S, XU C S, LI Y C. Advance in biosynthesis of lignin and its regulation. Acta Botanica Boreali-Occidentalia Sinica, 2003, 23(1): 171-181. (in Chinese)
[9]	XIE M, ZHANG J, TSCHAPLINSKI T J, TUSKAN G A, CHEN J G, MUCHERO W. Regulation of lignin biosynthesis and its role in growth-defense tradeoffs. Frontiers in Plant Science, 2018, 9: 1427. doi: 10.3389/fpls.2018.01427 pmid: 30323825
[10]	CESARINO I. Structural features and regulation of lignin deposited upon biotic and abiotic stresses. Current Opinion in Biotechnology, 2019, 56: 209-214. doi: S0958-1669(18)30062-4 pmid: 30684783
[11]	EYNCK C, SÉGUIN-SWARTZ G, CLARKE W E, PARKIN A P. Monolignol biosynthesis is associated with resistance to Sclerotinia sclerotiorum in Camelina sativa. Molecular Plant Pathology, 2012, 13(8): 887-899. doi: 10.1111/mpp.2012.13.issue-8
[12]	CHEZEM W R, MEMON A, LI F S, WENG J K, CLAY N K. SG2-Type R2R3-MYB transcription factor MYB15 controls defense- induced lignification and basal immunity in Arabidopsis. The Plant Cell, 2017, 29(8): 1907-1926. doi: 10.1105/tpc.16.00954
[13]	LI C, HE Q L, ZHANG F, YU J W, LI C, ZHAO T L, ZHANG Y, XIE Q W, SU B R, MEI L, ZHU S J, CHEN J H. Melatonin enhances cotton immunity to Verticillium wilt via manipulating lignin and gossypol biosynthesis. The Plant Journal, 2019, 100(4): 784-800. doi: 10.1111/tpj.v100.4
[14]	TANG Y, ZHANG Z N, LEI Y, HU G, LIU J F, HAO M Y, CHEN A M, PENG Q Z, WU J H. Cotton WATS modulate SA biosynthesis and local lignin deposition participating in plant resistance against Verticillium dahliae. Frontiers in Plant Science, 2019, 10: 526. doi: 10.3389/fpls.2019.00526
[15]	金贺. 大豆胞囊线虫胁迫下微紫青霉处理对GmC4H和GmCAD的影响及抗性机制研究[D]. 沈阳: 沈阳农业大学, 2022.
	JIN H. Effects of Penicillium purpureus on GmC4H and GmCAD under soybean cyst nematode stress and their resistance mechanism[D]. Shenyang: Shenyang Agricultural University, 2022. (in Chinese)
[16]	COHEN Y, EYAL H, HANANIA J. Ultrastructure, autofluorescence, callose deposition and lignification in susceptible and resistant muskmelon leaves infected with the powdery mildew fungus Sphaerotheca fuliginea. Physiological and Molecular Plant Pathology, 1990, 36(3): 191-204. doi: 10.1016/0885-5765(90)90025-S
[17]	PREISNER M, WOJTASIK W, KOSTYN K, BOBA A, CZUJ T, SZOPA J, KULMA A. The cinnamyl alcohol dehydrogenase family in flax: Differentiation during plant growth and under stress conditions. Journal of Plant Physiology, 2018, 221: 132-143. doi: S0176-1617(17)30304-8 pmid: 29277026
[18]	BORGES M F, RESENDE M L V, PINHO R G V. Time of inoculation and inoculum concentration in relation to the expression of corn stalk resistance to Fusarium moniliforme. Fitopatologia Brasileira, 2001, 26(4): 715-720. doi: 10.1590/S0100-41582001000400004
[19]	GAYOSO C, POMAR F, NOVO-UZAL E, MERINO F, DE ILÁRDUYA O M. The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H₂O₂, peroxidase and lignins and drives PAL gene expression. BMC Plant Biology, 2010, 10(1): 232. doi: 10.1186/1471-2229-10-232
[20]	XU L, ZHU L F, TU L L, LIU L L, YUAN D J, JIN L, LONG L, ZHANG X L. Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry. Journal of Experimental Botany, 2011, 62(15): 5607-5621. doi: 10.1093/jxb/err245 pmid: 21862479
[21]	XIAO S H, HU Q, SHEN J L, LIU S M, YANG Z G, CHEN K, KLOSTERMAN S J, JAVORNIK B, ZHANG X L, ZHU L F. GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae. Plant Cell Reports, 2021, 40(4): 735-751. doi: 10.1007/s00299-021-02672-x
[22]	XIONG X P, SUN S C, ZHU Q H, ZHANG X Y, LI Y J, LIU F, XUE F, SUN J. The cotton lignin biosynthetic gene Gh4CL30 regulates lignification and phenolic content and contributes to Verticillium wilt resistance. Molecular Plant-Microbe Interactions, 2021, 34 (3): 240-254. doi: 10.1094/MPMI-03-20-0071-R
[23]	KIM S J, KIM K W, CHO M H, FRANCESCHI V R, DAVIN L B, LEWIS N G. Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: Lessons for database annotations? Phytochemistry, 2007, 68 (14): 1957-1974.
[24]	SABALLOS A, EJETA G, SANCHEZ E, KANG C, VERMERRIS W. A genomewide analysis of the cinnamyl alcohol dehydrogenase family in Sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the brown midrib6 gene. Genetics, 2009, 181(2): 783-795. doi: 10.1534/genetics.108.098996
[25]	SIBOUT R, EUDES A, MOUILLE G, POLLTE B, LAPIERRE C, JOUANIN L, SÉGUIN A. CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. The Plant Cell, 2005, 17(7): 2059-2076. doi: 10.1105/tpc.105.030767
[26]	LIU W, JIANG Y, WANG C H, ZHAO L L, JIN Y Z, XING Q J, LI M, LV T H, QI H Y. Lignin synthesized by CmCAD2 and CmCAD3 in oriental melon (Cucumis melo L.)seedlings contributes to drought tolerance. Plant Molecular Biology, 2020, 103(6): 689-704. doi: 10.1007/s11103-020-01018-7
[27]	RONG W, LUO M Y, SHAN T L, WEI X N, DU L P, XU H J, ZHANG Z Y. A wheat cinnamyl alcohol dehydrogenase TaCAD12 contributes to host resistance to the sharp eyespot disease. Frontiers in Plant Science, 2016, 7: 1723. pmid: 27899932
[28]	TRONCHET M, BALAGUÉ C, KROJ T, JOUANIN L, ROBY D. Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Molecular Plant Pathology, 2010, 11(1): 83-92. doi: 10.1111/mpp.2010.11.issue-1
[29]	LIU L, WANG D, ZHANG C, LIU H Y, GUO H M, CHENG H M, LIU E L, SU X F. The heat shock factor GhHSFA4a positively regulates cotton resistance to Verticillium dahliae. Frontiers in Plant Science, 2022, 13: 1050216. doi: 10.3389/fpls.2022.1050216
[30]	陈敏, 林元秘, 朱文姣, 杨清. 茄子miR171b在抵抗黄萎病菌侵染中的功能. 农业生物技术学报, 2022, 30(7): 1268-1278.
	CHEN M, LIN Y M, ZHU W J, YANG Q. Function of eggplant (Solanum melongena) miR171b in resistance to Verticillium dahliae infection. Journal of Agricultural Biotechnology, 2022, 30(7): 1268-1278. (in Chinese)
[31]	LIU S P, ZHU Y P, XIE C, JUE D W, HONG Y B, CHEN M, HUBDAR A K, YANG Q. Transgenic potato plants expressing StoVe1 exhibit enhanced resistance to Verticillium dahliae. Plant Molecular Biology Reporter, 2012, 30(4): 1032-1039. doi: 10.1007/s11105-012-0413-y
[32]	SUN Y, WU Y E, ZHAO Y, HAN X J, LOU H X, CHENG A X. Molecular cloning and biochemical characterization of two cinnamyl alcohol dehydrogenases from a liverwort Plagiochasma appendiculatum. Plant Physiology and Biochemistry, 2013, 70: 133-141. doi: 10.1016/j.plaphy.2013.05.027
[33]	王卓, 徐碧玉, 贾彩红, 李健平, 刘菊华, 张建斌, 苗红霞, 金志强. 香蕉肉桂醇脱氢酶基因的克隆及表达分析. 西北植物学报, 2015, 35(7): 1305-1310.
	WANG Z, XU B Y, JIA C H, LI J P, LIU J H, ZHANG J B, MIAO H X, JIN Z Q. Molecular cloning and expression of cinnamyl alcohol dehydrogenase gene from banana. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(7): 1305-1310. (in Chinese)
[34]	KIM S J, KIM M R, BEDGAR D L, MOINUDDIN S G, CARDENAS C L, DAVIN L B, KANG C, LEWIS N G. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(6): 1455-1460.
[35]	TOBIAS C M, CHOW E K. Structure of the cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification. Planta, 2005, 220(5): 678-688. pmid: 15452707
[36]	SABALLOS A, EJETA G, SANCHEZ E, KANG C, VERMERRIS W. A genomewide analysis of the cinnamyl alcohol dehydrogenase family in Sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the brown midrib6 gene. Genetics, 2009, 181(2): 783-795. doi: 10.1534/genetics.108.098996
[37]	BARAKAT A, BAGNIEWSKA-ZADWORNA A, CHOI A, PLAKKAT U, DILORETO D S, YELLANKI P, CARLSON J. The cinnamyl alcohol dehydrogenase gene family in Populus: Phylogeny, organization, and expression. BMC Plant Biology, 2009, 9: 26. doi: 10.1186/1471-2229-9-26
[38]	LI H P, ZHANG S L, ZHAO Y L, ZHAO X L, XIE W F, GAO Y T, WANG Y G, LI K, GUO J G, ZHU Q H, ZHANG X B, JIA K P, MIAO Y C. Identification and characterization of cinnamyl alcohol dehydrogenase encoding genes involved in lignin biosynthesis and resistance to Verticillium dahliae in upland cotton (Gossypium hirsutum L.). Frontiers in Plant Science, 2022, 13: 840397. doi: 10.3389/fpls.2022.840397
[39]	MORANT M, HEHN A, WERCK-REICHHART D. Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants. BMC Plant Biology, 2002, 2: 7. pmid: 12153706
[40]	SAIDI M N, BOUAZIZ D, HAMMAMI I, NAMSI A, DRIRA N, GARGOURI-BOUZID R. Alterations in lignin content and phenylpropanoids pathway in date palm (Phoenix dactylifera L.) tissues affected by brittle leaf disease. Plant Science, 2013, 211: 8-16. doi: 10.1016/j.plantsci.2013.06.008
[41]	TRONCHET M, BALAGUÉC, KROJ T, JOUANIN L, ROBY D. Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Molecular Plant Pathology, 2010, 11(1): 83-92. doi: 10.1111/mpp.2010.11.issue-1
[42]	BHUIYAN N H, SELVARAJ G, WEI Y D, KING J. Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion. Journal of Experimental Botany, 2009, 60(2): 509-521. doi: 10.1093/jxb/ern290 pmid: 19039100
[43]	段雅婕, 杨宝明, 郭志祥, 尹可锁, 胡会刚, 曾莉, 白亭亭. 外源水杨酸诱导香蕉苯丙烷类代谢提高对枯萎病抗性. 热带作物学报, 2022, 43(9): 1870-1879.
	DUAN Y J, YANG B M, GUO Z X, YIN K S, HU H G, ZENG L, BAI T T. Exogenous salicylic acid induced phenylpropane metabolism in banana to improve the resistance against Fusarium wilt. Chinese Journal of Tropical Crops, 2022, 43(9): 1870-1879. (in Chinese)
[44]	李婷婷. 疫霉菌Avr3a家族效应蛋白保守靶标CAD7的鉴定与功能研究[D]. 杨凌: 西北农林科技大学, 2017.
	LI T T. Identification and functional analysis of conserved target CAD7 by Phytophthora Avr3a family effectors[D]. Yangling: Northwest Agricultural and Forestry University, 2017. (in Chinese)