‘黑比诺’葡萄不同叶龄叶片叶绿体内淀粉积累及其相关基因表达差异分析

doi:10.3864/j.issn.0578-1752.2022.21.013

摘要/Abstract

摘要：

【目的】随着叶龄的增长，葡萄叶片叶绿体内淀粉积累增加，但是调控其淀粉积累的分子机理还未见报道，本研究通过筛选参与调控淀粉积累的潜在基因，阐明葡萄不同叶龄叶片叶绿体中淀粉积累的分子机理。【方法】以2年生‘黑比诺’(Pinot Noir)葡萄为试材，观察不同叶龄叶片叶绿体内淀粉积累动态，并采用高通量测序技术进行转录组分析，筛选出与淀粉和蔗糖代谢途径相关的关键酶基因，同时采用实时荧光定量PCR技术对关键基因进行表达验证。【结果】未展开叶（NL）叶绿体内淀粉粒体积较小，积累量少，随着叶龄增长，在生长中叶（GL）和成熟叶（ML）叶绿体内，淀粉粒体积明显增大，积累量增加。转录组测序共得到58.57 GB的有效数据，KEGG富集分析表明，与NL相比，在GL和ML中差异表达的基因显著富集于淀粉与蔗糖代谢通路。分析该通路基因表达模式发现，细胞壁转化酶（cell wall invertase,CWINV）、磷酸葡萄糖变位酶（Phosphoglucomutase，PGM）、腺苷二磷酸葡萄糖焦磷酸化酶（ADP-glucose pyrophosphorylase，AGPase）等基因参与蔗糖转化为淀粉合成前体腺苷二磷酸葡萄糖（adenosine diphosphate glucose，ADPG）的过程，且随叶龄增大逐渐上调表达;可溶性淀粉合成酶（soluble starch synthase，SSⅠ）、淀粉分支酶（starch branching enzyme，SBE）、α-淀粉酶（α-amylase，AMY）、β-淀粉酶（β-amylase，BAM）等基因参与淀粉合成与水解过程，同样随叶龄增大上调表达，其中AGPase、SSⅠ、SBE在半结晶结构支链淀粉合成中起重要作用。【结论】随着叶龄的增大，叶绿体中淀粉积累增加;AGPase、SSⅠ、SBE等基因可能是参与调控叶绿体内淀粉积累的关键基因。

关键词: 黑比诺, 转录组, 叶龄, 淀粉, 淀粉代谢

Abstract:

【Objective】The accumulation of starch in chloroplast of grape leaves increased with leaf maturation, but the related molecular mechanism has not been reported. In this study, the potential related genes were screened in order to clarify the molecular regulation mechanism of starch accumulation in chloroplast of grape leaves with different leaf ages.【Method】 Two years old ‘Pinot Noir’ grapes (Vitis vinifera L.) were used as plant materials to investigate the dynamic changes of starch accumulation in chloroplast of leaves with different ages. The RNA sequencing technology was used to characterize the differently expressed genes (DEGs) involved in starch and sucrose metabolism in leaves with different ages. The expression levels of several key candidate genes were detected by using real-time quantitative qPCR.【Result】The starch grains in NL (undeveloped leaves) were smallest in volume and lowest in number compared with these in GL (growing leaves) and ML (mature leaves). With the increase of leaf ages, the volume and amount of starch grains in GL and ML also increased. A total of 58.57 GB valid data was obtained by RNA-sequencing. Differentially expressed genes mainly involved in starch and sucrose metabolism pathway for the leaves with different ages. The genes involved in the process of sucrose conversion into starch synthesis precursor adenosine diphosphate glucose (ADP) showed up-regulated expression level with the increase of leaf ages, including cell wall invertase (CWINV), phosphoglucomutase (PGM), and ADP-glucose pyrophosphorylase (AGPase). The genes involved in starch synthesis and hydrolysis also presented up-regulated expression level with the increase of leaf ages, including soluble starch synthase (SSⅠ), starch branching enzyme (SBE), α-amylase (AMY), and β-amylase (BAM). AGPase, SSⅠ and SBE played an important role in synthesis of amylopectin with semi-crystalline structure.【Conclusion】With the increase of leaf ages, the starch accumulation in chloroplast also increased. AGPase, SSⅠ and SBE genes could be the key genes involved in the regulation of starch accumulation in chloroplast of grape leaves.

Key words: Pinot Noir, transcriptome, leaf age, starch, starch metabolism

尤佳玲,李有梅,孙孟豪,谢兆森. ‘黑比诺’葡萄不同叶龄叶片叶绿体内淀粉积累及其相关基因表达差异分析[J]. 中国农业科学, 2022, 55(21): 4265-4278.

YOU JiaLing,LI YouMei,SUN MengHao,XIE ZhaoSen. Analysis Reveals the Differential Expression of Genes Related to Starch Accumulation in Chloroplast of Leaf with Different Ages in Pinot Noir Grape[J]. Scientia Agricultura Sinica, 2022, 55(21): 4265-4278.

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参考文献 55

[1]	张芮, 成自勇, 王旺田, 吴玉霞, 牛黎莉, 张晓霞, 高阳, 陈娜娜, 马奇梅. 不同生育期水分胁迫对延后栽培葡萄产量与品质的影响. 农业工程学报, 2014, 30(24): 105-113.
	ZHANG R, CHENG Z Y, WANG W T, WU Y X, NIU L L, ZHANG X X, GAO Y, CHEN N N, MA Q M. Effect of water stress in different growth stages on grape yield and fruit quality under delayed cultivation facility. Transactions of the Chinese Society of Agricultural Engineering, 2014, 30(24): 105-113. (in Chinese)
[2]	IACONO F, SOMMER K J. Response of electron transport rate of water stress-affected grapevines: Influence of leaf age. Vitis, 2000, 39(4): 137-144.
[3]	CALUGR A, CORDEA M I, BABE A, FEJER M. Dynamics of starch reserves in some grapevine varieties (Vitis vinifera L.) during dormancy. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca Horticulture, 2019, 76(2): 185-192.
[4]	HUNTER J J, RUFFNER H P, VOLSCHENK C G. Starch concentrations in grapevine leaves, berries and roots and the effect of canopy management. South African Journal for Enology & Viticulture, 1995, 16(2): 35-40.
[5]	KIKUZAWA K, LECHOWICZ M J. Ecology of Leaf Longevity. Springer Tokyo, 2011: 23-35.
[6]	陶然. 葡萄果实发育阶段淀粉代谢分子机理与香气相关基因表达分析[D]. 南京: 南京农业大学, 2014.
	TAO R. Study on starch metabolism and expression analysis of aroma gene during grape berry development[D]. Nanjing: Nanjing Agricultural University, 2014. (in Chinese)
[7]	ZEEMAN S C, KOSSMANN J, SMITH A M. Starch: Its metabolism, evolution, and biotechnological modification in plants. Annual Review of Plant Biology, 2010, 61: 209-234. doi: 10.1146/annurev-arplant-042809-112301.
[8]	李肖蕖, 王建设, 张根发. 植物蔗糖转化酶及其基因表达调控研究进展. 园艺学报, 2008, 35(9): 1384-1392.
	LI X Q, WANG J S, ZHANG G F. Advanced in plant invertase and regulation of gene expression. Acta Horticulturae Sinica, 2008, 35(9): 1384-1392. (in Chinese)
[9]	HAYES M A, DAVIES C, DRY I B. Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: Differential roles in sink and source tissues. Journal of Experimental Botany, 2007, 58(8): 1985-1997. doi: 10.1093/jxb/erm061.
[10]	崔娜, 王卫平, 林凤, 白丽萍, 张玉龙. 植物果糖激酶的研究进展. 中国农学通报, 2010, 26(14): 41-47.
	CUI N, WANG W P, LIN F, BAI L P, ZHANG Y L. Update on fructokinase in higher plants. Chinese Agricultural Science Bulletin, 2010, 26(14): 41-47. (in Chinese)
[11]	李晓屿, 李玉花, 李晗, 李治龙, 蓝兴国. 植物葡萄糖磷酸变位酶的研究进展. 植物生理学报, 2015, 51(5): 617-622.
	LI X Y, LI Y H, LI H, LI Z L, LAN X G. Advances in research on phosphoglucomutase in plants. Plant Physiology Journal, 2015, 51(5): 617-622. (in Chinese)
[12]	BALLICORA M A, IGLESIAS A A, PREISS J. ADP-glucose pyrophosphorylase: A regulatory enzyme for plant starch synthesis. Photosynthesis Research, 2004, 79(1): 1-24. doi: 10.1023/b:pres.0000011916.67519.58.
[13]	苗红霞, 孙佩光, 张凯星, 金志强, 徐碧玉. 植物颗粒结合淀粉合成酶(GBSS)基因的表达调控机制研究进展. 生物技术通报, 2016, 32(3): 18-23.
	MIAO H X, SUN P G, ZHANG K X, JIN Z Q, XU B Y. Research progress on expression regulation mechanism of genes encoding granule-bound starch synthase in plants. Biotechnology Bulletin, 2016, 32(3): 18-23. (in Chinese)
[14]	张军杰, 黄玉碧. 玉米可溶性淀粉合成酶研究进展. 玉米科学, 2006, 14(6): 151-154.
	ZHANG J J, HUANG Y B. Resrarch progress in maize (Zea mays L.) soluble starch synthase. Journal of Maize Sciences, 2006, 14(6): 151-154. (in Chinese)
[15]	姚新灵, 丁向真, 陈彦云, 吴晓玲, 郭蔼光. 淀粉分支酶和去分支酶编码基因的功能. 植物生理学通讯, 2005, 41(2): 253-259.
	YAO X L, DING X Z, CHEN Y Y, WU X L, GUO A G. Functions of genes encoding starch branch enzyme and debranch enzyme. Plant Physiology Communications, 2005, 41(2): 253-259. (in Chinese)
[16]	ZHAO L Y, GONG X, GAO J Z, DONG H Z, ZHANG S L, TAO S T, HUANG X S. Transcriptomic and evolutionary analyses of white pear (Pyrus bretschneideri) β-amylase genes reveals their importance for cold and drought stress responses. Gene, 2019, 689: 102-113. doi: 10.1016/j.gene.2018.11.092.
[17]	WINKLER A J, WILLIAMS W O. Starch and sugars of Vitis vinifera. Plant Physiology, 1945, 20(3): 412-432. doi: 10.1104/pp.20.3.412.
[18]	ZHANG P A, LU S W, LIU Z J, ZHENG T, DONG T Y, JIN H C, JIA H F, FANG J G. Transcriptomic and metabolomic profiling reveals the effect of LED light quality on fruit ripening and anthocyanin accumulation in cabernet sauvignon grape. Frontiers in Nutrition, 2021, 8: 790697. doi: 10.3389/fnut.2021.790697.
[19]	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. 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, 2021, 21: 156-171.
[20]	贺安娜, 谭晓利. 虎耳草不同叶龄光合特性及叶片结构比较. 中国农学通报, 2011, 27(16): 122-125.
	HE A N, TAN X L. Comparative analysis of different leaf age of saxifrage based on photosynthetic characters and leaf structure. Chinese Agricultural Science Bulletin, 2011, 27(16): 122-125. (in Chinese)
[21]	周会萍, 王晓冰, 徐鑫, 周晓君. 红叶石楠不同叶龄叶片的光合特性研究. 西部林业科学, 2020, 49(1): 39-45.
	ZHOU H P, WANG X B, XU X, ZHOU X J. Photosynthetic characteristics of Photinia fraseri leaves at different ages. Journal of West China Forestry Science, 2020, 49(1): 39-45. (in Chinese)
[22]	理挪, 王培, PEGGY C A E, 林思祖, 陈宇. 不同叶龄杉木叶片形态及光合特性分析. 亚热带农业研究, 2018, 14(3): 167-171.
	LI N, WANG P, PEGGY C A E, LIN S Z, CHEN Y. Leaf morphology and photosynthetic characteristics at different leaf ages in Chinese fir. Subtropical Agriculture Research, 2018, 14(3): 167-171. (in Chinese)
[23]	吴桂成, 张洪程, 吴文革, 王艳, 戴其根, 霍中洋, 许轲, 魏海燕. 不同叶龄期追施穗肥对粳型超级稻产量及品质的影响. 安徽农业科学, 2010, 38(18): 9440-9441, 9528.
	WU G C, ZHANG H C, WU W G, WANG Y, DAI Q G, HUO Z Y, XU K, WEI H Y. Effects of dressing ear fertilizer in different leaf age on the yield and quality of Japonica super rice. Journal of Anhui Agricultural Sciences, 2010, 38(18): 9440-9441, 9528. (in Chinese)
[24]	刘慧迪, 杨克军, 李佐同, 王玉凤, 张翼飞, 王智慧, 付健, 谷英楠, 杨系玲, 吴琼. 松嫩平原西部膜下滴灌玉米基于叶龄指数的适宜追氮量研究. 植物营养与肥料学报, 2016, 22(3): 811-820.
	LIU H D, YANG K J, LI Z T, WANG Y F, ZHANG Y F, WANG Z H, FU J, GU Y N, YANG X L, WU Q. Suitable amount of N topdressing based on leaf age index of maize using mulched drip irrigation technology in Western Songnen Plain. Journal of Plant Nutrition and Fertilizer, 2016, 22(3): 811-820. (in Chinese)
[25]	史晓敏, 刘竞择, 张艳霞, 陈祖民, 郭帅奇, 王振平. 水分胁迫对‘赤霞珠’不同叶龄叶片光合特性的影响. 果树学报, 2021, 38(1): 50-63.
	SHI X M, LIU J Z, ZHANG Y X, CHEN Z M, GUO S Q, WANG Z P. Effects of water stress on photosynthetic characteristics of ‘Cabernet Sauvignon’ at different leaf ages. Journal of Fruit Science, 2021, 38(1): 50-63. (in Chinese)
[26]	郭帅奇, 刘竞择, 张艳霞, 陈祖民, 史晓敏, 王振平. 水分胁迫对‘赤霞珠’葡萄不同叶龄叶片糖含量及相关代谢酶活性的影响. 北方园艺, 2021(12): 17-26.
	GUO S Q, LIU J Z, ZHANG Y X, CHEN Z M, SHI X M, WANG Z P. Effects of water stress on sugar content and related metabolic enzyme activities in leaves of ‘Cabernet Sauvignon’ grape at different leaf ages. Northern Horticulture, 2021(12): 17-26. (in Chinese)
[27]	李予霞, 崔百明, 董新平, 王雪莲. 水分胁迫下葡萄叶片脯氨酸和可溶性总糖积累与叶龄的关系. 果树学报, 2004, 21(2): 170-172.
	LI Y X, CUI B M, DONG X P, WANG X L. Relationship between accumulation of proline and soluble sugar with age of grape leaves in water stress. Journal of Fruit Science, 2004, 21(2): 170-172. (in Chinese)
[28]	JAILLON O, AURY J M, NOEL B, POLICRITI A, CLEPET C, CASAGRANDE A, CHOISNE N, AUBOURG S, VITULO N, JUBIN C, VEZZI A, LEGEAI F, HUGUENEY P, DASILVA C, HORNER D, MICA E, JUBLOT D, POULAIN J, BRUYÈRE C, BILLAULT A, et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature, 2007, 449(7161): 463-467. doi: 10.1038/nature06148.
[29]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-△△CT method. Methods, 2001, 25(4): 402-408. doi: 10.1006/meth.2001.1262.
[30]	张懿, 张大兵, 刘曼. 植物体内糖分子的长距离运输及其分子机制. 植物学报, 2015, 50(1): 107-121. doi: 10.3724/SP.J.1259.2015.00107.
	ZHANG Y, ZHANG D B, LIU M. The molecular mechanism of long-distance sugar transport in plants. Chinese Bulletin of Botany, 2015, 50(1): 107-121. doi: 10.3724/SP.J.1259.2015.00107. (in Chinese)
[31]	李金亭, 胡正海, 高鹏. 木立芦荟不同叶龄叶的解剖学和组织化学及其植物化学研究. 西北植物学报, 2007, 27(11): 2202-2209.
	LI J T, HU Z H, GAO P. Anatomy,histochemistry and phytochemistry of Aloe arborescens leaves according to leaf-age. Acta Botanica Boreali-Occidentalia Sinica, 2007, 27(11): 2202-2209. (in Chinese)
[32]	张娟, 王玉安. 施氮量对‘马瑟兰’葡萄叶片糖代谢的影响. 甘肃农业大学学报, 2020, 55(6): 97-103.
	ZHANG J, WANG Y A. Effects of nitrogen application on glucose metabolism in ‘Maseran’ grape leaves. Journal of Gansu Agricultural University, 2020, 55(6): 97-103. (in Chinese)
[33]	李鹏程, 李铭, 郁松林, 郭绍杰, 苏学德. GA₃对葡萄果实淀粉积累及代谢相关酶活性的影响. 北方园艺, 2011(11): 17-22.
	LI P C, LI M, YU S L, GUO S J, SU X D. Effects of GA₃ on the amylopectin accumulation and related enzyme activities in amylopectin metabolism of grape fruits. Northern Horticulture, 2011(11): 17-22. (in Chinese)
[34]	刘敏, 房玉林. 高温胁迫对葡萄幼树生理指标和超显微结构的影响. 中国农业科学, 2020, 53(7): 1444-1458.
	LIU M, FANG Y L. Effects of heat stress on physiological indexes and ultrastructure of grapevines. Scientia Agricultura Sinica, 2020, 53(7): 1444-1458. (in Chinese)
[35]	张洁, 李天来. 日光温室亚高温对番茄光合作用及叶绿体超微结构的影响. 园艺学报, 2005, 32(4): 614-619.
	ZHANG J, LI T L. Effects of daytime sub-high temperature on photosynthesis and chloroplast ultrastructure of tomato leaves in greenhouse. Acta Horticulturae Sinica, 2005, 32(4): 614-619. (in Chinese)
[36]	马晓丽, 刘雪峰, 杨梅, 颜秋杨, 袁项成, 向苹苇. 镁肥对葡萄叶片糖、淀粉和蛋白质及果实品质的影响. 中国土壤与肥料, 2018(4): 114-120.
	MA X L, LIU X F, YANG M, YAN Q Y, YUAN X C, XIANG P W. Effects of magnesium application on the leaves sugar, starch and protein content and the fruit quality of grapes. Soil and Fertilizer Sciences in China, 2018(4): 114-120. (in Chinese)
[37]	ZIMMERMANN P, HIRSCH-HOFFMANN M, HENNIG L, GRUISSEM W. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiology, 2004, 136(1): 2621-2632. doi: 10.1104/pp.104.046367.
[38]	KEURENTJES J J, SULPICE R, GIBON Y, STEINHAUSER M C, FU J, KOORNNEEF M, STITT M, VREUGDENHIL D. Integrative analyses of genetic variation in enzyme activities of primary carbohydrate metabolism reveal distinct modes of regulation in Arabidopsis thaliana. Plant Signaling & Behavior, 2008, 9(8): R129. doi: 10.1186/gb-2008-9-8-r129.
[39]	LAFTA A M, FUGATE K K. Metabolic profile of wound-induced changes in primary carbon metabolism in sugarbeet root. Phytochemistry, 2011, 72(6): 476-489. doi: 10.1016/j.phytochem.2010.12.016.
[40]	MALINOVA I, KUNZ H H, ALSEEKH S, HERBST K, FERNIE A R, GIERTH M, FETTKE J, KUSANO M. Reduction of the cytosolic phosphoglucomutase in Arabidopsis reveals impact on plant growth, seed and root development, and carbohydrate partitioning. PLoS ONE, 2014, 9(11): e112468.
[41]	林雪茜, 彭淼, 吴少平, 易干军, 董涛, 钟晓红, 高慧君. ‘中蕉9号’与‘巴西蕉’果实后熟过程中可溶性糖积累差异的原因分析. 果树学报, 2019, 36(11): 1524-1539.
	LIN X X, PENG M, WU S P, YI G J, DONG T, ZHONG X H, GAO H J. A comparative analysis of the differences in starch degradation and soluble sugar accumulation between ‘Zhongjiao No. 9’ and ‘Baxijiao’ during fruit ripening. Journal of Fruit Science, 2019, 36(11): 1524-1539. (in Chinese)
[42]	余春梅, 陈佩度, 季本华. 小麦胚乳淀粉合成酶基因研究进展. 麦类作物学报, 2004, 24(4): 123-128.
	YU C M, CHEN P D, JI B H. Advances on the genes of starch synthesis enzymes in wheat endosperm. Acta Tritical Crops, 2004, 24(4): 123-128. (in Chinese)
[43]	VENTRIGLIA T, KUHN M L, RUIZ M T, RIBEIRO-PEDRO M, VALVERDE F, BALLICORA M A, PREISS J, ROMERO J M. Two Arabidopsis ADP-Glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic. Plant Physiology, 2008, 148(1): 65-76.
[44]	BOEHLEIN S K, SHAW J R, BOEHLEIN T J, BOEHLEIN E C, HANNAH L C. Fundamental differences in starch synthesis in the maize leaf, embryo, ovary and endosperm. The Plant Journal, 2018, 96(3): 595-606. doi: 10.1111/tpj.14053.
[45]	BULÉON A, COLONNA P, PLANCHOT V, BALL S. Starch granules: Structure and biosynthesis. International Journal of Biological Macromolecules, 1998, 23(2): 85-112.
[46]	DELVALLÉ D, DUMEZ S, WATTEBLED F, ROLDÁN I, PLANCHOT V, BERBEZY P, COLONNA P, VYAS D, CHATTERJEE M, BALL S, MÉRIDA A, D'HULST C. Soluble starch synthase I: A major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves. The Plant Journal, 2005, 43(3): 398-412. doi: 10.1111/j.1365-313x.2005.02462.x.
[47]	SZYDLOWSKI N, RAGEL P, HENNEN-BIERWAGEN T A, PLANCHOT V, MYERS A M, MÉRIDA A, D'HULST C, WATTEBLED F. Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch. Journal of Experimental Botany, 2011, 62(13): 4547-4559. doi: 10.1093/jxb/err172.
[48]	陈雅玲, 包劲松. 水稻胚乳淀粉合成相关酶的结构、功能及其互作研究进展. 中国水稻科学, 2017, 31(1): 1-12.
	CHEN Y L, BAO J S. Progress in structures, functions and interactions of starch synthesis related enzymes in rice endosperm. Chinese Journal of Rice Science, 2017, 31(1): 1-12. (in Chinese)
[49]	NISHI A, NAKAMURA Y, TANAKA N, SATOH H. Biochemical and genetic analysis of the effects of Amylose-Extender mutation in rice endosperm. Plant Physiology, 2001, 127(2): 459-472.
[50]	KASEMSUWAN T, JANE J L, SCHNABLE P, ROBERTSON D. Characterization of the dominant mutant amylose-extender (Ae1-5180) maize starch. Cereal Chemistry, 1995, 72(5): 457-464.
[51]	SMITH A M, NEUHAUS H E, STITT M. The impact of decreased activity of starch-branching enzyme on photosynthetic starch synthesis in leaves of wrinkled-seeded peas. Planta, 1990, 181(3): 310-315. doi: 10.1007/BF00195881.
[52]	YU T S, ZEEMAN S C, THORNEYCROFT D, FULTON D C, DUNSTAN H, LUE W L, HEGEMANN B, TUNG S Y, UMEMOTO T, CHAPPLE A, TSAI D L, WANG S M, SMITH A M, CHEN J, SMITH S M. Alpha-Amylase is not required for breakdown of transitory starch in Arabidopsis leaves. The Journal of Biological Chemistry, 2005, 280(11): 9773-9779. doi: 10.1074/jbc.m413638200.
[53]	王海波, 王森, 何平, 常源升, 李林光, 何晓文. 苹果β-淀粉酶基因MdBAM3的克隆和低温响应表达分析. 分子植物育种, 2020, 18(13): 4205-4212.
	WANG H B, WANG S, HE P, CHANG Y S, LI L G, HE X W. Cloning and expression response to low-temperature stress of β-amylase gene (MdBAM3) in apple. Molecular Plant Breeding, 2020, 18(13): 4205-4212. (in Chinese)
[54]	PENG T, ZHU X F, DUAN N, LIU J H. 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, 2014, 37(12): 2754-2767. doi: 10.1111/pce.12384.
[55]	周凯悦. 大豆盐胁迫下叶绿体淀粉积累转录组及相关基因功能研究[D]. 杭州: 浙江大学, 2020.
	ZHOU K Y. Transcriptome analysis and research of related gene function of starch accumu lation in soybean chloroplast under salt stress[D]. Hangzhou: Zhejiang University, 2020. (in Chinese)

基因名称 Name	基因ID Gene ID	正向引物 Forward primer sequence (5′-3′)	反向引物 Reverse primer sequence (5′-3′)
VvActin7	VIT_04s0044g00580	F: CTTGCATCCCTCAGCACCTT	R: TCCTGTGGACAATGGATGGA
VvCWINV1	VIT_09s0002g02320	F: TTGACAACGCTAAGAACCGAAGAA	R: GAATTGCCTGAACTCCAGACCATC
VvHXK2	VIT_09s0002g03390	F: GAGCACTACAGTGAGTACAGTAAG	R: GAATGAGAGGCAGCAAGAAGG
VvFRK1	VIT_15s0048g01260	F: AAGTCTGGTAGCATCCTCTCTTAT	R: TCAGCCTGGTTCCATATACTCAT
VvFRK2	VIT_05s0102g00710	F: GCTGTAGAGGGATTTCATGTCAA	R: CTCTCAATCTTGCCTCGTCTTC
VvFRK4	VIT_18s0089g01230	F: GCATCAAGAGCATCTGGAACC	R: ACCATCAGTGACCACAAGCA
VvPGM	VIT_01s0011g05370	F: GCAACCATCCGTGTCTATATTGA	R: TTGAGAGCAACCTCCACAAGA
VvAGPS1.2	VIT_03s0038g04570	F: GTGCCATTCTATGCCATACTGAT	R: GAATCCGCTACTGGTGGTGTA
VvSBEⅠ	VIT_08s0007g03750	F: GGAGACAGAATAACCAAGATGACA	R: GCTGGCAAGATGATACCTTACAA
VvSBEⅡ	VIT_18s0001g00060	F: GCTGAGCAGGAGAGTCTTGA	R: GTAGCCTCTTCCACCACATCT
VvAMY1.1	VIT_03s0063g00400	F: CAACCTTAACGGCTTCTCCAAT	R: ATGAGTGATTCCAGAGGCAGATA
VvAMY1.2	VIT_03s0063g00450	F: AGCAAGACGCAAGAGGAATATG	R: GCACTGTAGTCATCTCCAGAATC
VvAMY2	VIT_18s0001g00560	F: GCCACCAGCATCTCAGTCT	R: ACTATGTCAGCCATTGCTCTAAC
VvBAM2	VIT_15s0046g02620	F: TTGTGATGGAGGCAAGTATGATAG	R: TGGCTAGAGCAAGTACACGAT
VvBAM3	VIT_02s0012g00170	F: GGCTGAATTAACTGCTGGATACTA	R: TTGCTGCTCCCTGTCCTTC
VvSSⅠ	VIT_16s0098g01780	F: TAGTGCCAGTGCTTTTGGCT	R: CACAGCCCCATACCACTCAG

样品名称 Sample name	质控后的 clean reads数 Total reads	比对到参考基因组上的reads数及百分比 Total map reads (%)	比对到唯一位置的reads数及百分比 Unique map reads (%)	比对到多个位置的reads数及百分比 Multi map reads (%)	比对到参考基因组正链上的reads数及百分比 Positive map reads (%)	比对到参考基因组负链上的reads数及百分比 Negative map reads (%)
NL1	43198062	39524089 (91.5%)	38617667 (89.4%)	906422 (2.1%)	19289169 (44.65%)	19328498 (44.74%)
NL2	42652276	39262139 (92.05%)	38352259 (89.92%)	909880 (2.13%)	19157641 (44.92%)	19194618 (45.0%)
NL3	41550992	38222716 (91.99%)	37345353 (89.88%)	877363 (2.11%)	18653976 (44.89%)	18691377 (44.98%)
GL1	43373766	39810586 (91.78%)	38866791 (89.61%)	943795 (2.18%)	19454902 (44.85%)	19411889 (44.75%)
GL2	45964018	42408116 (92.26%)	41440537 (90.16%)	967579 (2.11%)	20735166 (45.11%)	20705371 (45.05%)
GL3	42560230	39115832 (91.91%)	38227440 (89.82%)	888392 (2.09%)	19122008 (44.93%)	19105432 (44.89%)
ML1	44057236	40379107 (91.65%)	39408268 (89.45%)	970839 (2.2%)	19690278 (44.69%)	19717990 (44.76%)
ML2	44139002	40538462 (91.84%)	39625981 (89.78%)	912481 (2.07%)	19802759 (44.86%)	19823222 (44.91%)
ML3	43029030	39519670 (91.84%)	38610003 (89.73%)	909667 (2.11%)	19297553 (44.85%)	19312450 (44.88%)

基因ID Gene ID	基因名称 Gene name	FPKM			GL vs NL		ML vs GL		ML vs NL
基因ID Gene ID	基因名称 Gene name	NL	GL	ML	Log2FC	P_adj	Log2FC	P_adj	Log2FC	P_adj
VIT_09s0002g02320	CWINV	9.46	28.66	32.22	1.59	2.91E-09	0.13	6.37E-01	1.77	1.12E-16
VIT_09s0002g03390	HXK2	1.19	0.54	0.00	-1.13	3.32E-02	-6.25	2.60E-05	-7.33	4.42E-08
VIT_15s0048g01260	FRK1	5.74	3.21	0.58	-0.84	7.46E-03	-2.52	1.18E-08	-3.32	3.60E-17
VIT_05s0102g00710	FRK2	79.24	57.48	21.44	-0.47	1.14E-04	-1.46	1.60E-31	-1.88	6.07E-43
VIT_18s0089g01230	FRK4	7.45	9.78	0.15	0.39	3.40E-01	-6.10	1.32E-25	-5.67	8.66E-24
VIT_01s0011g05370	PGM	59.83	68.55	149.43	0.19	2.68E-01	1.09	6.58E-16	1.32	4.67E-36
VIT_03s0038g04570	AGPS1.2	1.63	1.97	50.92	0.27	4.68E-01	4.66	1.56E-17	4.97	1.15E-17
VIT_16s0098g01780	SSⅠ	12.37	64.56	151.46	2.38	6.35E-86	1.19	1.79E-41	3.62	6.82E-223
VIT_08s0007g03750	SBEⅠ	10.74	18.68	75.62	0.79	1.67E-05	1.98	4.39E-91	2.82	5.34E-63
VIT_18s0001g00060	SBEⅡ	46.07	53.83	96.15	0.22	1.17E-01	0.80	7.37E-12	1.06	1.02E-35
VIT_03s0063g00400	AMY1.1	0.95	5.34	10.63	2.49	6.30E-10	0.95	1.26E-03	3.49	1.92E-41
VIT_03s0063g00450	AMY1.2	0.27	1.21	1.09	2.15	3.67E-03	-0.19	8.04E-01	2.00	2.26E-02
VIT_18s0001g00560	AMY2	5.33	9.79	19.47	0.87	3.78E-07	0.95	5.26E-11	1.87	7.50E-33
VIT_15s0046g02620	BAM2	10.80	17.79	33.68	0.71	2.63E-07	0.88	1.50E-14	1.64	2.38E-42
VIT_02s0012g00170	BAM3	1.19	26.15	93.39	4.45	7.37E-38	1.80	2.47E-14	6.29	1.27E-147