毛花猕猴桃AeBAM基因家族鉴定及其表达分析

doi:10.16420/j.issn.0513-353x.2025-0310

摘要/Abstract

摘要：

为解析毛花猕猴桃（Actinidia eriantha）β-淀粉酶（β-amylase，BAM）基因家族信息，基于毛花猕猴桃‘赣绿1号’高质量参考基因组数据（PRJNA839193），鉴定了AeBAM家族成员并对其理化性质、进化关系进行了分析，同时利用转录组和qRT-PCR技术明确了AeBAM在不同组织及果实发育后期（淀粉降解期）的表达特征。在毛花猕猴桃中共鉴定出17个AeBAM成员，编码氨基酸数量在189 ~ 751之间、理论等电点5.24 ~ 10.01、分子量21 149.85 ~ 84 142.08 Da。根据多物种进化树分析，BAM可以分为5个亚族，其中I和V亚族成员居多；共线性分析表明，AeBAM家族内部共存在10对复制基因对和5对串联重复基因对，所有成员的K_a/K_s比值均小于1，在进化过程中受到纯化选择；AeBAM家族基因启动子上含有大量的光响应、逆境胁迫和激素调控相关的顺式作用元件。转录组分析表明，AeBAM成员表达具有组织特异性，其中AeBAM5、AeBAM13和AeBAM16在果实中具有较高的表达量且在果实套袋后表达量显著上升；qRT-PCR表明，AeBAM5和AeBAM13表达量在淀粉降解期显著上升并与淀粉降解负相关，可被认为是调控果实淀粉降解的关键基因家族成员。

关键词: 毛花猕猴桃, β-淀粉酶, 淀粉, 表达分析

Abstract:

To elucidate the characteristics of the β-amylase（BAM）gene family in Actinidia eriantha，AeBAM family members were identified based on high-quality reference genome of A. eriantha‘Ganlü 1’（PRJNA839193），and their physicochemical properties and evolutionary relationships were analyzed. In addition，transcriptome sequencing and qRT-PCR analyses were performed to determine the expression profiles of AeBAM genes in different tissues and at the late fruit development（starch degradation stage）. A total of seventeen AeBAM genes were identified in A. eriantha，encoding proteins ranging from 189 to 751 amino acids，with theoretical isoelectric points of 5.24-10.01 and molecular weights of 21 149.85-84 142.08 Da. Phylogenetic analysis across many species classified BAM into five subgroups，with subgroups I and V containing the majority members. Collinearity analysis revealed 10 pairs of duplicated gene pairs and 5 pairs of tandemly duplicated genes pairs within the family. All members showed K_a/K_s ratios less than 1，indicating they were purifying selection during evolution. Promoter analysis indicated that AeBAM genes contained numerous cis-acting elements related to light responsiveness，stress adaptation，and hormonal regulation. Transcriptome analysis showed that AeBAM genes exhibited tissue-specific expression patterns，among which AeBAM5，AeBAM13 and AeBAM16 were highly expressed in fruits，and their expression levels significant increased after bagging. qRT-PCR validation revealed that the expression levels of AeBAM5 and AeBAM13 increased significantly during the starch degradation stage and were negatively correlated with starch degradation，suggesting that they may be key members of the AeBAM gene family regulating fruit starch degradation.

Key words: Actinidia eriantha, β-amylase, starch, expression analysis

陈钰珊, 高欢, 吴俊康, 王莉梅, 黄春辉, 徐小彪, 廖光联. 毛花猕猴桃AeBAM基因家族鉴定及其表达分析[J]. 园艺学报, 2026, 53(3): 683-696.

CHEN Yushan, GAO Huan, WU Junkang, WANG Limei, HUANG Chunhui, XU Xiaobiao, LIAO Guanglian. Identification and Expression Analysis of AeBAM Gene Family in Actinidia eriantha[J]. Acta Horticulturae Sinica, 2026, 53(3): 683-696.

图/表 9

表1 qRT-PCR引物序列

Table 1 qRT-PCR primer sequences

基因 Gene	上游引物序列（5′-3′） Forward primer sequences	下游引物序列（5′-3′） Revers primer sequences	产物大小/bp Product
AeBAM1	GAATGGCTACAAGAGGCT	TATGTCGGGATTGCTACG	157
AeBAM2	GGAGGAAGGCTATGTGGG	TCCGTGTAGGCAAGGTCA	283
AeBAM3	TTTGTGCCCACTTTATCC	CAACCTCTTGTAGCCATT	384
AeBAM4	TACGCTATCCCTCTTATCC	ATTTCTGCCGCTTTCTTC	109
AeBAM5	TTTCCTTGGGTTGTGATT	GTCTTTCTTTCCGATTGC	281
AeBAM6	AGGGATGTTGCTTCTTCA	GGTTCATCTGCTGCTCATT	266
AeBAM7	CCCGATGGCACTACCTAT	GCACCCTGGAACCAACAT	298
AeBAM8	GATTCACTGGCACTACGGG	GGGCATCCTGAGGCTGTT	173
AeBAM9	GCCGATTGATGGAGTAAG	GATGCGTGGAAGCAGAGC	264
AeBAM10	GGCAAGGTGGTGTCAGTC	CATCCGATTTCCAGTTTT	103
AeBAM11	AGAAGGGAAGCCTCAGTG	GTTAGTTCAGCAGGGTGT	297
AeBAM12	TCAAGGATTGGCTGTGGC	GATGACGGCTGTTGATGC	141
AeBAM13	AGTGCGGAGGAAATGTTGG	TCTAAGCACTGGTAACGAAT	158
AeBAM14	GTGCGGAGGAAATGTTGG	AAGGATAGGTATTCGGCATT	129
AeBAM15	AGTGCGGAGGAAATGTTGG	GCCAAGGATAGGTATTCGGG	133
AeBAM16	TGGAACAATGATTACGGACAA	GTAGTGCCAATGAATCCCTG	150
AeBAM17	ATCTGATTCCAGGGTGCC	CATTCATTGTTCGTGGCTTG	89

表2 毛花猕猴桃AeBAM成员编码蛋白的理化性质

Table 2 Protein physical and chemical properties analysis of AeBAM

基因名 Gene name	基因ID Gene ID	氨基酸数 No. of amino acid	分子量/Da Molecular weight	等电点 pI	稳定系数 Instability index	脂溶指数 Aliphatic index	亲水性 Hydrop- athicity	已报道同源基因 Reported homologous genes
AeBAM1	Chr02G799	751	84 142.08	5.82	34.45	77.75	-0.35	/
AeBAM2	Chr02G1164	568	63 467.87	5.57	40.59	68.89	-0.45	/
AeBAM3	Chr06G622	700	78 501.26	5.24	38.18	73.40	-0.46	/
AeBAM4	Chr06G623	556	63 133.23	5.65	37.67	74.69	-0.39	/
AeBAM5	Chr11G331	480	54 004.19	8.29	33.44	67.25	-0.53	AdBAM3L（Acc28966）； AcBAM5（Actinidia10870.t1）
AeBAM6	Chr11G561	541	60 915.04	6.65	37.90	67.80	-0.31	/
AeBAM7	Chr11G638	666	74 694.92	5.89	39.40	70.33	-0.45	/
AeBAM8	Chr13G233	564	62 786.18	5.90	45.97	70.27	-0.42	/
AeBAM9	Chr16G812	532	58 600.29	6.18	28.35	81.73	-0.26	AcBAM13（Actinidia00051.t1）
AeBAM10	Chr16G1012	189	21 149.85	10.01	44.29	111.43	0.17	/
AeBAM11	Chr19G603	523	59 697.34	8.93	51.50	80.38	-0.30	/
AeBAM12	Chr20G1070	645	72 789.04	6.20	41.02	71.21	-0.48	/
AeBAM13	Chr22G622	547	61 739.99	8.62	38.39	66.54	-0.53	AdBAM3.1（Acc15874）
AeBAM14	Chr23G1204	501	56 841.28	8.24	37.43	67.19	-0.58	/
AeBAM15	Chr23G1205	301	34 345.80	8.96	40.58	82.23	-0.34	/
AeBAM16	Chr23G1206	501	56 428.79	8.00	37.67	66.99	-0.56	/
AeBAM17	Chr25G384	546	61 462.78	8.58	36.62	70.57	-0.51	AdBAM3（Acc16695）

图1 AeBAM基因家族的系统发育、保守域和基因结构分析

Fig. 1 Phylogenetic，conserved domains and gene structure analysis of the AeBAM gene family

图2 AeBAM家族基因共线性与Ka/Ks分析 A：AeBAM家族成员内部共线性分析；B：AeBAM家族成员Ka/Ks分析；C：多物种BAM家族成员间共线性分析

Fig. 2 Collinearity and Ka/Ks analysis of AeBAM gene family members A：Collinearity analysis among AeBAM family members；B：Ka/Ks analysis of AeBAM family members；C：Collinearity analysis of BAM family members across multiple species

图3 毛花猕猴桃和多个物种的BAM家族基因系统进化树分析 Ac：中华猕猴桃；Ae：毛花猕猴桃；Al：阔叶猕猴桃；Ar：山梨猕猴桃；At：拟南芥；；Md：苹果；Os：水稻；Pb：梨；Vv：葡萄

Fig. 3 Phylogenetic tree analysis of BAM between Actinidia eriantha and other species Ac：Actinidia chinensis；Ae：Actinidia eriantha；Al：Actinidia latifolia；Ar：Actinidia rufa；At：Arabidopsis thaliana；Md：Malus domestica；Os：Oryza sativa；Pb：Pyrus bretschneideri；Vv：Vitis vinifera

图4 毛花猕猴桃AeBAM家族基因启动子顺式作用元件分析括号中为各顺式元件的数量

Fig. 4 cis-Acting elements analysis of AeBAM gene promoter Numbers in parentheses indicate the count of each cis-acting element

图5 毛花猕猴桃AeBAM组织表达特性

Fig. 5 Tissues expression analysis of AeBAMs

图6 毛花猕猴桃果实套袋后AeBAM家族基因的表达量 * 表示在P < 0.05水平具有显著性差异，** 表示在P < 0.01水平具有显著性差异，*** 表示在P < 0.001水平具有显著性差异，**** 表示在P < 0.0001水平具有显著性差异，ns 表示无显著差异

Fig. 6 Expression analysis of AeBAM family genes after fruit bagging * indicates significant differences at P < 0.05，** indicates significant differences at P < 0.01，*** indicates significant differences at P < 0.001，**** indicates significant differences at P < 0.0001，ns indicates no significant difference

图7 毛花猕猴桃AeBAM家族基因在果实发育后期的相对表达量分析

Fig. 7 Relative expression analysis of AeBAM family genes during the late fruit development

参考文献 33

[1]	Bailey T L, Johnson J, Grant C E, Noble W S. 2015. The MEME Suite. Nucleic Acids Research, 43 (1),39-49.
[2]	Chen C J, Wu Y, Li J W, Wang X, Zeng Z H, Xu J, Liu Y L, Feng J T, Chen H, He Y H, Xia R. 2023. TBtools-II:A“One for All,All for One”bioinformatics platform for biological big-data mining. Molecular Plant, 16 (11),1733-1742. doi: 10.1016/j.molp.2023.09.010 URL
[3]	Chen Mindong, Wang Bin, Bai Changhui, Qiu Boyin, Liu Jianding, Wen Qingfang, Zhu Haisheng, Li Yongping. 2025. Identification of expansin family gene and its expression analysis during fruit enlargement in sponge gourd(Luffa cylindrica). Acta Horticulturae Sinica,2025, 52 (6):1488-1504. (in Chinese) doi: 10.16420/j.issn.0513-353x.2024-0373
	陈敏氡, 王彬, 白昌辉, 裘波音, 刘建汀, 温庆放, 朱海生, 李永平. 2025. 普通丝瓜Expansin家族基因的鉴定及其在果实膨大中的表达分析. 园艺学报,2025, 52 (6):1488-1504. doi: 10.16420/j.issn.0513-353x.2024-0373
[4]	Fan X L, Lin H R, Ding F, Ling W M. 2024. Jasmonates promote β-amylase-mediated starch degradation to confer cold tolerance in tomato plants. Plants(Basel,Switzerland), 13 (8),1055.
[5]	Fuentes-Beals C, Valdés-Jiménez A, Riadi G. 2022. Hidden Markov Modeling with HMMTeacher. PLoS Computational Biology, 18 (2):e1009703. doi: 10.1371/journal.pcbi.1009703 URL
[6]	Fulton D C, Stettler M, Mettler T, Vaughan C K, Li J, Francisco P, Gil M, Reinhold H, Eicke S, Messerli G L, Dorken G, Halliday K, Smith A M, Smith S M, Zeeman S C. 2008. β-Amylase4,a noncatalytic protein required for starch break down,acts upstream of three active β-amylases in Arabidopsis chloroplasts. The Plant Cell, 20 (4):1040-1058. doi: 10.1105/tpc.107.056507 URL
[7]	Gong X C, Lin M F, Song J, Mao J P, Yao D L, Gao Z, Wang X L. 2025. Genome-wide identification of the AcBAM family in kiwifruit(Actinidia chinensis cv. Hongyang)and the expression profiling analysis of AcBAMs reveal their role in starch metabolism. BMC Plant Biology,25,415.
[8]	Hao Xinyuan, Yue Chuan, Tang Hu, Qian Wenjun, Wang Yuchun, Wang Lu, Wang Xinchao, Yang Yajun. 2017. Cloning of β-amylase gene(CsBAM3)and its expression model response to cold stress in tea plant. Acta Agronomica Sinica, 43 (10),1417-1425. (in Chinese) doi: 10.3724/SP.J.1006.2017.01417
	郝心愿, 岳川, 唐湖, 钱文俊, 王玉春, 王璐, 王新超, 杨亚军. 2017. 茶树β-淀粉酶基因CsBAM3的克隆及其响应低温的表达模式. 作物学报, 43 (10):1417-1425. doi: 10.3724/SP.J.1006.2017.01417
[9]	Huang D, Wang X, Tang Z, Yuan Y, Xu Y, He J, Jiang X, Peng S A, Li L, Butelli E, Deng X, Xu Q. 2018. Subfunctionalization of the Ruby2-Ruby 1 gene cluster during the domestication of citrus. Nature Plants, 4 (11),930-941. doi: 10.1038/s41477-018-0287-6 pmid: 30374094
[10]	Jia D F, Liao G L, Ye B, Zhong M, Huang C H, Xu X B. 2024. Changes in fruit quality,phenolic compounds,and antioxidant activity of kiwifruit (Actinidia eriantha)during on-vine ripening. LWT-Food Science and Technology,206:116564.
[11]	Lao N T, Schoneveld O, Mould R M, Hibberd J M, Gray J C, Kavanagh T A. 1999. An Arabidopsis gene encoding a chloroplast-targeted β-amylase. The Plant Journal, 20 (5):519-527. doi: 10.1046/j.1365-313X.1999.00625.x URL
[12]	Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Peer Y V D, Rouzé P, Rombauts S. 2002. PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research, 30 (1):325-327.
[13]	Letunic I, Bork P. 2024. Interactive Tree of Life(iTOL)v6:recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Research, 52 (1):78-82.
[14]	Li Y Q, Gao H, Jia D F, Wang H L, Zheng K X, Xu X B. 2024. AGPS and BAM genes dramatically improve‘Ganlv 1’(Actinidia eriantha)fruit quality through starch metabolism during fruit development. Scientia Horticulturae,239:113004.
[15]	Liang G P, He H H, Nai G J, Feng L D, Li Y M, Zhou Q, Ma Z H, Yue Y, Chen B H, Mao J. 2021. Genome-wide identification of BAM genes in grapevine(Vitis vinifera L.) and ectopic expression of VvBAM1 modulating soluble sugar levels to improve low-temperature tolerance in tomato. BMC Plant Biology, 21 (1):156. doi: 10.1186/s12870-021-02916-8
[16]	Liao G L, Huang C H, Jia D F, Zhong M, Tao J J, Qu X Y, Xu X B. 2023. High quality genome of Actinidia eriantha provide new insight into ascorbic acid regulation. Journal of Integrative Agriculture, 22 (11):3244-3255. doi: 10.1016/j.jia.2023.07.018 URL
[17]	Liao G L, Xu X B, Huang C H, Zhong M, Jia D F. 2021. Resource evaluation and novel germplasm mining of Actinidia eriantha. Scientia Horticulturae,282:110037.
[18]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-∆∆CT method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[19]	Ma Y P, Han Y R, Feng X R, Gao H D, Cao B, Song L H. 2022. Genome-wide identification of BAM(β-amylase)gene family in jujube(Ziziphus jujuba Mill.)and expression in response to abiotic stress. BMC Genomics, 23 (1):438. doi: 10.1186/s12864-022-08630-5
[20]	Monroe J D, Storm A R. 2018. Review:the Arabidopsis β-amylase(BAM)gene family:diversity of form and function. Plant Science,276:163-170.
[21]	Monroe J D, Storm A R, Badley E M, Lehman M D, Platt S M, Saunders L K, Schmitz J M, Torres C E. 2014. β-Amylase1 and β-amylase 3 are plastidic starch hydrolases in Arabidopsis that seem to be adapted for different thermal,pH,and stress condition. Plant Physiology, 166 (4):1748-1763. doi: 10.1104/pp.114.246421 URL
[22]	Niu L J, Wu X L, Liu H, Hu X L, Wang W. 2024. Leaf starch degradation by β-amylase ZmBAM8 influences drought tolerance in maize. Carbohydrate Polymers,345:122555.
[23]	Peng T, Zhu X F, Duan N, Liu J H. 2014. PtrBAM1,a β-amylase-coding gene of Poncirus trifoliata,is a CBF regulon member with function in cold tolerance by modulating soluble sugar levels. Plant,Cell & Environment, 37 (12):2754-2767. doi: 10.1111/pce.2014.37.issue-12 URL
[24]	Reinhold H, Soyk S, Šimková K, Hostettler C, Marafino J, Mainiero S, Vaughan C K, Monroe J D, Zeeman S C. 2011. β-Amylase-like proteins function as transcription factors in Arabidopsis,controlling shoot growth and development. The Plant Cell, 23 (4):1391-1403. doi: 10.1105/tpc.110.081950 pmid: 21487098
[25]	Shi Q B, Xia Y, Xue N, Wang Q B, Tao Q, Li M J, Xu D, Wang X F, Kong F Y, Zhang H S, Li G. 2024. Modulation of starch synthesis in Arabidopsis via phytochrome B-mediated light signal transduction. Journal of Integrative Plant Biology, 66 (5):973-985. doi: 10.1111/jipb.v66.5 URL
[26]	Smith A M, Zeeman S C, Smith S M. 2005. Starch degradation. Annual Review of Plant Biology,56:73-98.
[27]	Stitt M, Zeeman S C. 2012. Starch turnover:pathways,regulation and role in growth. Current Opinion in Plant Biology, 15 (3):282-292. doi: 10.1016/j.pbi.2012.03.016 pmid: 22541711
[28]	Tian Xiaocheng, Zhu Lingcheng, Zou Hui, Li Baiyun, Ma Fengwang, Li Mingjun. 2023. Research progress on accumulation pattern and regulation of soluble sugar in fruit. Acta Agronomica Sinica, 50 (4):885-895. (in Chinese)
	田晓成, 祝令成, 邹晖, 李白云, 马锋旺, 李明军. 2023. 果实可溶性糖的积累模式及其调控研究进展. 园艺学报, 50 (4):885-895. doi: 10.16420/j.issn.0513-353x.2021-1264
[29]	Tong Y, Su W Y, Chen Y T, Liu X F, Zhang Q Y, Qi T H, Allan A C, Li X, Yin X R. 2024. Two ethylene-responsive transcription factors,AdVAL2 and AdKAN2,regulate early steps in kiwifruit starch degradation. Postharvest Biology and Technology,216:113058.
[30]	Wang Cheng. 2023. Effect of low temperature on starch metabolism and sugar accumulation in oriental melon(Cucumis melo var. makuwa Makino)during fruit enlarging and postharvest[M. D. Dissertation]. Shenyang:Shenyang Agricultural University. (in Chinese)
	王成. 2023. 低温对薄皮甜瓜果实膨大期和采后淀粉代谢和糖积累的影响[硕士论文]. 沈阳:沈阳农业大学.
[31]	Wang Xinru, Bai Shuaishuai, Sun Yule, Wu Xinyu, Zeng Feng, Liu Chunying, Zhang Yuxi, Gai Shupeng, Yuan Yanchao. 2025. Genome-wide identification of PoSnRK gene family in Paeonia ostii and expression analysis during flower senescence. Acta Horticulturae Sinica, 52 (9):2363-2376. (in Chinese) doi: 10.16420/j.issn.0513-353x.2024-0993
	王心茹, 白帅帅, 孙雨乐, 武欣宇, 曾凤, 刘春英, 张玉喜, 盖树鹏, 袁延超. 2025. ‘凤丹’牡丹PoSnRK基因家族鉴定及其在花衰老中的表达分析. 园艺学报, 52 (9):2363-2376. doi: 10.16420/j.issn.0513-353x.2024-0993
[32]	Yao X H, Wang S B, Wang Z P, Li D W, Jiang Q, Zhang Q, Gao L, Zhong C H, Huang H W, Liu Y F. 2022. The genome sequencing and comparative analysis of a wild kiwifruit Actinidia eriantha. Molecular Horticulture, 2 (1):13. doi: 10.1186/s43897-022-00034-z
[33]	Zhao Liangyi. 2019. Bioinformatics analysis of pear β-amylase family and identification of PbBAM3 gene cold resistance[M. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	赵粱怡. 2019. 梨β-淀粉酶基因家族生物信息学分析及PbBAM3基因抗寒性鉴定[硕士论文]. 南京: 南京农业大学.