柚MYB基因家族的鉴定及其在砧穗不亲和叶片黄化中的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0873

摘要/Abstract

摘要：

目的柑橘主要依赖嫁接繁殖，但常因砧穗不亲和导致经济损失。探究MYB基因家族成员在柑橘砧穗不亲和黄化叶片中的作用，为深入解析柑橘砧穗互作机制奠定基础。方法利用基因组对柚（Citrus grandis）MYB基因家族成员进行全基因组鉴定与系统分析；基于亲和/不亲和砧穗组合不同黄化阶段叶片转录组分析和荧光定量检测，筛选关键MYB基因；通过烟草叶片瞬时过表达验证候选基因CgMYB53在砧穗不亲和叶片黄化中的功能。结果在柚基因组中共鉴定出143个MYB基因家族成员，分为R1-MYB（35个）、R2R3-MYB（104个）、R3-MYB（3个）和R4-MYB（1个）4个亚家族。MYB基因启动子序列主要包含光响应（53.23%）、激素响应（27.94%）和胁迫响应（16.07%）等元件。通过比较亲和/不亲和砧穗组合的叶片转录组，筛选到10个差异表达的MYB基因。实时荧光定量结果显示，CgMYB53在正常叶片中相对表达量较低，在黄化叶片中表达量较高。烟草叶片中瞬时过表达CgMYB53，可导致叶绿素a和叶绿素b含量极显著下降，而叶片中直链淀粉、支链淀粉和总淀粉含量极显著升高，淀粉合成基因CgGBSS2、CgISA2和CgBE3表达量极显著上升。结论在柚中鉴定出143个MYB基因家族成员，筛选出10个可能在砧穗不亲和黄化叶片中起调控作用的MYB基因。CgMYB53可能调控淀粉合成途径基因表达促使淀粉堆积进而参与叶片黄化。

关键词: 柑橘, 砧穗亲和性, 淀粉代谢, 叶绿素降解, 全基因组分析, MYB基因家族, 启动子分析, 瞬时过表达

Abstract:

Objective Grafting is commonly used in citrus propagation, while scion-rootstock incompatibility often occurs in orchards and leads to economic losses. This study is aimed to investigate the role of MYB family genes in leaf chlorosis associated with scion-rootstock incompatibility, and to elucidate the molecular mechanisms underlying scion-rootstock interaction in Citrus. Method We performed a genome-wide identification and systematic analysis of the MYB gene family in pummelo (Citrus grandis). Based on transcriptome data and RT-qPCR from leaves at different yellowing stages of compatible/incompatible graft combinations, key MYB genes were screened. By employing transient overexpression in tobacco leaves, the function of the candidate gene CgMYB53 in leaf chlorosis associated with scion-rootstock incompatibility was preliminarily validated. Result A total of 143 MYB family members were identified in the pomelo genome, classified into four subfamilies, R1-MYB (35 members), R2R3-MYB (104 members), R3-MYB (3 members), and R4-MYB (1 member). Promoter sequences of MYB members primarily contained cis-acting elements responsive to light (53.23%), hormones (27.94%), and stress (16.07%). Comparative transcriptome analysis of leaves from compatible and incompatible graft combinations, 10 differentially expressed MYB genes were identified. RT-qPCR results showed that the relative expression of CgMYB53 was relatively low in the green leaves, but relatively high in the yellow leaves. Transient overexpression of CgMYB53 in tobacco leaves resulted in significant decrease in chlorophyll a and chlorophyll b content, accompanied by significant increase in amylose, amylopectin, and total starch content,starch synthesis-related genes CgGBSS2、CgISA2 and CgBE3 were highly expressed. Conclusion This study identified 143 MYB family members in pomelo and screened 10 MYB TFs potentially involved in leaf chlorosis associated with scion-rootstock incompatibility. Preliminary functional validation indicates that CgMYB53 may participates in starch synthesis related genes expression, inducing leaf chlorosis by regulating starch accumulation.

Key words: citrus, graft compatibility, starch metabolism, chlorophyll degradation, genome-wide analysis, MYB family gene, promoter analysis, transient overexpression

郭道祥, 苏全, 李奥婷, 王燕, 陈清, 王小蓉, 何文. 柚MYB基因家族的鉴定及其在砧穗不亲和叶片黄化中的功能分析[J]. 生物技术通报, 2026, 42(4): 170-181.

GUO Dao-xiang, SU Quan, LI Ao-ting, WANG Yan, CHEN Qing, WANG Xiao-rong, HE Wen. Identification of the MYB Gene Family in Pomelo (Citrus grandis) and Functional Analysis in Chlorosis of Graft Incompatibility[J]. Biotechnology Bulletin, 2026, 42(4): 170-181.

图/表 9

表1 RT-qPCR、基因克隆和质粒构建引物序列

Table 1 Primer sequences for RT-qPCR, gene cloning, and plasmid construction

基因 Gene	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	退火温度 Annealing temperature （℃）	产物长度 Primer length （bp）
β-Tubulin	ACATCCCGCCTAAGGGTCTG	TTCCTCCGAAACATAGCCGTA	58	150
q-CgAPL8	ATTGGAGCCCTTCTCTCAGC	AACTGCTCTGTCTCAGCCAG	58	150
q-CgISA2	CCATTACTACACGGCTCTTCT	GCGATTGAGGCTTCTTGATAA	58	150
q-CgISA3	GGCATTGGGTGACTGAGTTT	CCCATCTCTTCCAATGAGGA	58	150
q-CgMYB53	GAGGCACATTCCCAAAGCTG	TGTTCGTCCTGGAAGTCTCC	58	150
q-Cg2g041650	TGGGAGCTCCCAAGTAAGGC	GGCTTATCAGTCCCCGTTGC	58	150
q-CgGBSS2	TAGCCGCTTCAAGCTTTGTCT	CAATCATTAAGGATGGCCCGTTCT	58	150
q-MYB45	TCATGGTGAGGGCAATTGGG	CAATGACCATCGGTTGCCAA	58	150
q-MYB75	ATTCCCAGCAGAGGCACTAG	CCACGCTCTCAAACACAACG	58	150
q-MYB40	ACTCGAAGAGCGACCCATAA	TATCGTTCGACAAGCCTGGT	58	150
q-MYB113	AGAGGACCAGCTATTGCTGA	ATGATCTCGTCTTCGTCGGG	58	150
q-MYB136	AAGCACAGAAAGGGCTTGTG	AATCCATCTCAGTCTGCAGC	58	150
q-MYB129	CGCCCACATCAAAAAACACG	ATCCATCGGAGTCTGCAACT	58	150
q-MYB2	TCGAGTTGGACAGCCAAGAA	CAACGAGCATCTCATACCGC	58	150
q-MYB89	GAATCAAGTTCTGGCCTGCA	TTGGCTTGGGAGCAAAATCG	58	150
q-MYB14	CTTCGAAAGGGCTTGTGGTC	ACAGCTCTTGCCACATCTCT	58	150
q-CgBE3	GACCGTCAACTCCCCTCATA	GCTGATCAACCCTTGGAAAA	58	150
q-CgBE1	ACTTCGCTTCCTTCTGTCCA	CCAGAAACATCTTCGGCAAT	58	150
q-CgAPL3	TCTTAAGAGCGGGGACTTGA	CCACATTTTTGGGATCTGC	58	150
q-CgBAM6	GCTGTTGCAGAGATGGTTGA	GAAGATCATCAACGGCCAAT	58	150
q-CgBAM4	TCTCCGGGCTAAGAGTTCAA	CCAGTGGCAACATCACAAAC	58	150
CgMYB53	ATGGCGGGTAAGCGCAAGA	CAAAATCCCAAATCCGAT	60	753
OE-CgMYB53	GAACACGGGGGACGAGCTCGGTACCATGGCGGGTAAGCGCAAGA	GTGGTGGTGGTCGACGGATCCTCACCAAAATCCCAAATCCGAT	62	799

图1 CgMYBs染色体定位

Fig. 1 Chromosomal localization of CgMYBs

图2 CgMYBs基因的物种内共线性分析A：CgMYBs基因的种内共线性分析；B：存在共线性的MYB基因对在不同砧穗组合中的表达热图分析［Log10（FPKM+0.01）］；亲和砧穗组合：GxPt（枳砧‘琯溪蜜柚’）和HmCj（‘蜀砧1号’砧‘红绵蜜柚’）；不亲和砧穗组合：HmPt（枳砧‘红绵蜜柚’）；S2：相对叶绿素含量（SPAD值）范围为70‒80；S4：SPAD值范围为50‒60；S8：SPAD值范围为10‒20。下同

Fig. 2 Co-linearity analysis of CgMYBs genesA: Co-linearity analysis of CgMYBs genes. B: Expression heatmap of genes involved in co-linearity CgMYBs in different graft combinations. Compatible graft combinations: GxPt (‘Guanxi Miyou’ grafted onto trifoliate orange) and HmCj (‘Hongmian Miyou’ grafted onto ‘Shuzhen No.1’). Incompatible graft combination: HmPt (‘Hongmian Miyou’ grafted onto trifoliate orange). S2: Relative chlorophyll content [Soil and Plant Analyzer Development (SPAD) value] between 70-80; S4: SPAD value between 50-60; S8: SPAD value between 10-20. The same below

图3 R2R3-CgMYBs基因启动子元件检测结果A：R2R3-CgMYBs不同类别的启动子元件数量；B：R2R3-CgMYBs激素响应元件所包含的启动子元件；C：R2R3-CgMYBs胁迫响应元件所包含的启动子元件；D：R2R3-CgMYBs植物生长相关响应元件所包含的启动子元件

Fig. 3 Assay result of promoter elements of the R2R3-CgMYBs genesA: Numbers of various promoter elements of the R2R3-CgMYBs. B: Promotor elements in hormone responsive elements of the R2R3-CgMYBs. C: Promotor elements in stress responsive elements of the R2R3-CgMYBs. D: Promotor elements in plant growth-related responsive elements of the R2R3-CgMYBs

图4 R2R3-CgMYBs成员系统进化关系（A）、保守基序（B）和基因结构分析（C）

Fig. 4 Phylogenetic tree (A), conserved motif (B), and gene structures (C) of R2R3-CgMYBs members

图5 R2R3-AtMYBs与R2R3-CgMYBs系统发育树

Fig. 5 Phylogenetic tree of R2R3-AtMYBs and R2R3-CgMYBs

图6 柚MYB基因在不同砧穗组合中的表达模式分析A：MYB成员在枳砧‘红绵蜜柚’（不亲和砧穗组合）叶片不同黄化阶段差异表达Venn分析；B：筛选出的10个CgMYBs在不同砧穗组合中的表达热图分析［Log10（FPKM+0.01）］

Fig. 6 Expression profile analysis of genes involved in CgMYBs in different graft combinationsA: Venn analysis of MYB genes in chlorotic phase of HmPt (incompatible graft). B: Expression heatmap of the ten CgMYBs in different graft combinations [Log10(FPKM+0.01)]

图7 关键MYB成员在HmPt 8个黄化阶段中的表达情况S1： SPAD 80‒90. S2： SPAD 70‒80. S3： SPAD 60‒70. S4： SPAD 50‒60. S5： SPAD 40‒50. S6： SPAD 30‒40. S7： SPAD 20‒30. S8： SPAD 10‒20

Fig. 7 Expression pattern of MYB in the leaves of HmPt at eight etiolation states

图8 烟草叶片瞬时过表达CgMYB53A：瞬时过表达CgMYB53烟草叶片表型及碘-碘化钾染色；B：CgMYB53相对表达量；C：烟草叶片中淀粉含量；D：烟草叶片中叶绿素含量（ND表示未检测到）；E：淀粉代谢途径相关基因表达情况；* P<0.05；** P<0.01；*** P<0.001

Fig. 8 Transient overexpressions of CgMYB53 in tobacco leavesA: Phenotype and iodine-potassium iodide staining of CgMYB53-transient overexpression tobacco leaves. B: Relative expressions of CgMYB53. C: Starch content in tobacco leaves. D: Chlorophyll and carotenoid content in tobacco leaves (ND stands for not detected). E: Relative expressions of starch metabolism-related genes. * P<0.05; ** P<0.01; *** P<0.001

参考文献 43

[1]	邓秀新. 中国柑橘育种60年回顾与展望 [J]. 园艺学报, 2022, 49(10): 2063-2074.
	Deng XX. A review and perspective for citrus breeding in China during the last six decades [J]. Acta Hortic Sin, 2022, 49(10): 2063-2074.
[2]	朱世平, 陈传武, 刘兆俊, 等. 卡里佐枳橙在中国的生产适应性调查与比较研究 [J]. 中国南方果树, 2020, 49(3): 1-8.
	Zhu SP, Chen CW, Liu ZJ, et al. Investigation and comparison of the performance of carrizo citrange in China [J]. South China Fruits, 2020, 49(3): 1-8.
[3]	He W, Xie R, Wang Y, et al. Comparative transcriptomic analysis on compatible/incompatible grafts in citrus [J]. Hortic Res, 2022, 9: uhab072.
[4]	He W, Wang Y, Chen Q, et al. Dissection of the mechanism for compatible and incompatible graft combinations of Citrus grandis (L.) Osbeck (‘Hongmian Miyou’) [J]. Int J Mol Sci, 2018, 19(2): 505.
[5]	He W, Xie R, Guo DX, et al. The starch excess and key genes underlying citrus leaf chlorosis by rootstock-scion incompatibility [J]. Int J Biol Macromol, 2024, 282(Pt 4): 137111.
[6]	He W, Xie R, Luo L, et al. Comparative transcriptomic analysis of inarching invigorating rootstock onto incompatible grafts in citrus [J]. Int J Mol Sci, 2022, 23(23): 14523.
[7]	Zhang LC, Zhao GY, Jia JZ, et al. Molecular characterization of 60 isolated wheat MYB genes and analysis of their expression during abiotic stress [J]. J Exp Bot, 2012, 63(1): 203-214.
[8]	Karpinska B, Karlsson M, Srivastava M, et al. MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen [J]. Plant Mol Biol, 2004, 56(2): 255-270.
[9]	Lim PO, Kim HJ, Nam HG. Leaf senescence [J]. Annu Rev Plant Biol, 2007, 58: 115-136.
[10]	Guo YF, Ren GD, Zhang KW, et al. Leaf senescence: progression, regulation, and application [J]. Mol Hortic, 2021, 1(1): 5.
[11]	Zhang YM, Guo PR, Xia XL, et al. Multiple layers of regulation on leaf senescence: new advances and perspectives [J]. Front Plant Sci, 2021, 12: 788996.
[12]	Yongfeng Guo SG. AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis [J]. Plant Physiol, 2011, 156(3): 1612-1619.
[13]	Zhang X, Ju HW, Chung MS, et al. The R-R-type MYB-like transcription factor, AtMYBL, is involved in promoting leaf senescence and modulates an abiotic stress response in Arabidopsis [J]. Plant Cell Physiol, 2011, 52(1): 138-148.
[14]	Buelbuel S, Sakuraba Y, Sedaghatmehr M, et al. Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence [J]. Plant J, 2023, 116(1): 251-268.
[15]	Wang WB, He XF, Yan XM, et al. Chromosome-scale genome assembly and insights into the metabolome and gene regulation of leaf color transition in an important oak species, Quercus dentata [J]. New Phytol, 2023, 238(5): 2016-2032.
[16]	Cao J, Yang Q, Zhao YN, et al. MYB47 delays leaf senescence by modulating jasmonate pathway via direct regulation of CYP94B3/CYP94C1 expression in Arabidopsis [J]. New Phytol, 2025, 246(5): 2192-2206.
[17]	He SC, Zhi F, Min YC, et al. The MYB59 transcription factor negatively regulates salicylic acid- and jasmonic acid-mediated leaf senescence [J]. Plant Physiol, 2023, 192(1): 488-503.
[18]	Yang JW, Huang JZ, Wu X, et al. NtMYB1 and NtNCED1/2 control abscisic acid biosynthesis and tepal senescence in Chinese Narcissus (Narcissus tazetta) [J]. J Exp Bot, 2023, 74(21): 6505-6521.
[19]	Zhu ZF, Lu LL, Chen SY, et al. Transcription factor CitMYB16 positively regulates cutin and wax biosynthesis in citrus by directly activating CitDCR and CitKCS2 [J]. Plant J, 2025, 123(3): e70324.
[20]	Liu CY, Long JM, Zhu KJ, et al. Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis [J]. Sci Rep, 2016, 6: 25352.
[21]	Wang TT, Song X, Zhang M, et al. CsCPC, an R3-MYB transcription factor, acts as a negative regulator of citric acid accumulation in Citrus [J]. Plant J, 2025, 121(1): e17189.
[22]	Liu H, Wang X, Liu SJ, et al. Citrus Pan-Genome to Breeding Database (CPBD): a comprehensive genome database for citrus breeding [J]. Mol Plant, 2022, 15(10): 1503-1505.
[23]	Mistry J, Chuguransky S, Williams L, et al. Pfam: The protein families database in 2021 [J]. Nucleic Acids Res, 2021, 49(D1): D412-D419.
[24]	Lescot M, Déhais P, Thijs G, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences [J]. Nucleic Acids Res, 2002, 30(1): 325-327.
[25]	Kumar S, Stecher G, Li M, et al. MEGA X: Molecular evolutionary genetics analysis across computing platforms [J]. Mol Biol Evol, 2018, 35(6): 1547-1549.
[26]	唐云, 闫海彦, 赵亚雄, 等. 碘比色法测定高粱中直链淀粉和支链淀粉的方法探讨 [J]. 食品工业科技, 2023, 44(13): 272-280.
	Tang Y, Yan HY, Zhao YX, et al. Determination of amylose and amylopectin in sorghum by iodine colorimetric method [J]. Sci Technol Food Ind, 2023, 44(13): 272-280.
[27]	Wellburn AR, Lichtenthaler H. Formulae and program to determine total carotenoids and chlorophylls a and b of leaf extracts in different solvents [M]//Advances in Photosynthesis Research. Dordrecht: Springer Netherlands, 1984: 9-12.
[28]	Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis [J]. Trends Plant Sci, 2010, 15(10): 573-581.
[29]	Yang JH, Zhang BH, Gu G, et al. Genome-wide identification and expression analysis of the R2R3-MYB gene family in tobacco (Nicotiana tabacum L.) [J]. BMC Genomics, 2022, 23(1): 432.
[30]	Chen YH, Yang XY, He K, et al. The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family [J]. Plant Mol Biol, 2006, 60(1): 107-124.
[31]	Ding Y, Yang QH, Waheed A, et al. Genome-wide characterization and functional identification of MYB genes in Malus sieversii infected by Valsa mali [J]. Front Plant Sci, 2023, 14: 1112681.
[32]	Li XL, Xue C, Li JM, et al. Genome-wide identification, evolution and functional divergence of MYB transcription factors in Chinese white pear (Pyrus bretschneideri) [J]. Plant Cell Physiol, 2016, 57(4): 824-847.
[33]	Liu H, Xiong JS, Jiang YT, et al. Evolution of the R2R3-MYB gene family in six Rosaceae species and expression in woodland strawberry [J]. J Integr Agric, 2019, 18(12): 2753-2770.
[34]	Hou XJ, Li SB, Liu SR, et al. Genome-wide classification and evolutionary and expression analyses of citrus MYB transcription factor families in sweet orange [J]. PLoS One, 2014, 9(11): e112375.
[35]	Zhang DW, Zhou HP, Zhang Y, et al. Diverse roles of MYB transcription factors in plants [J]. J Integr Plant Biol, 2025, 67(3): 539-562.
[36]	Frangedakis E, Yelina NE, Billakurthi K, et al. MYB-related transcription factors control chloroplast biogenesis [J]. Cell, 2024, 187(18): 4859-4876.e22.
[37]	Li ZT, Gmitter FG, Grosser JW, et al. Isolation and characterization of a novel anthocyanin-promoting MYBA gene family in citrus [J]. Tree Genet Genomes, 2012, 8(4): 675-685.
[38]	Luo TT, Zhang H, Tan HK, et al. Two MYB transcription factors interact to inhibit the expression of cell wall metabolism and starch degradation genes in banana [J]. Plant Physiol, 2025, 198(4): kiaf239.
[39]	Xue J, Lu DB, Wang SG, et al. Integrated transcriptomic and metabolomic analysis provides insight into the regulation of leaf senescence in rice [J]. Sci Rep, 2021, 11(1): 14083.
[40]	Huang JY, Yan M, Zhu XY, et al. Gene mapping of starch accumulation and premature leaf senescence in the ossac3 mutant of rice [J]. Euphytica, 2018, 214(10): 177.
[41]	Huang KY, Chen L, Jiao GA, et al. OsMYBR1, a 1R-MYB family transcription factor regulates starch biosynthesis in rice endosperm [J]. Life, 2025, 15(6): 962.
[42]	Xiao QL, Wang YY, Du J, et al. ZmMYB14 is an important transcription factor involved in the regulation of the activity of the ZmBT1 promoter in starch biosynthesis in maize [J]. FEBS J, 2017, 284(18): 3079-3099.
[43]	Fan ZQ, Ba LJ, Shan W, et al. A banana R2R3-MYB transcription factor MaMYB3 is involved in fruit ripening through modulation of starch degradation by repressing starch degradation-related genes and MabHLH6 [J]. Plant J, 2018, 96(6): 1191-1205.