Physiobiochemical Changes and Gene Expressions of Polygonatum cyrtonema Hua Seeds undergone Post-ripening Treatment
-
摘要:
目的 探明多花黄精种子生理后熟过程中生理生化变化规律及差异基因表达情况。 方法 以多花黄精种子生理后熟过程6个阶段的种子为材料,采用化学测定方法,分析每个阶段的超氧化物歧化酶、α淀粉酶、β淀粉酶、可溶性蛋白、粗脂肪、可溶性糖含量。采用高通量转录组测序技术,对6个阶段的差异表达基因进行分析,并用qRT-PCR法对关键差异基因的表达量进行验证。 结果 (1)多花黄精种子生理后熟过程中,可溶性糖含量在前4个时期逐渐增高后,在后两个时期稍有下降;粗脂肪、蛋白质整体呈下降趋势;α淀粉酶活性及β淀粉酶活性呈现增强趋势。(2)多花黄精种子生理后熟过程中,蔗糖和淀粉代谢途径、激素信号转导通路等显著富集差异表达基因。 结论 多花黄精种子生理后熟过程中,淀粉等营养物质的代谢、转化与内源激素的协调作用,促进多花黄精种子的生理后熟。 Abstract:Objective Physiobiochemical changes and differentially expressed genes in Polygonatumcyrtonema Hua seeds after a post-ripening treatment were studied. Method Contents of superoxide dismutase, α-amylase, β-amylase, soluble protein, crude fat, and soluble sugar at 6 seed development stages were monitored. Differentially expressed genes were analyzed with high-throughput sequencing followed by a qRT-PCR verification. Result (1) The post-ripening treatment raised the soluble sugar content in the seeds gradually in the initial 4 stages and slightly declined in the last two stages, while induced a declining trend on the crude fat and protein. Meanwhile, the activities of α-amylase and β-amylase increased. (2) Significantly enriched differentially expressed genes were observed in the sucrose and starch metabolic pathways as well as the hormone signal transduction pathway during the process. Conclusion The post-ripening treatment on the P. cyrtonema seeds brought about the conversion and metabolism of starch and other nutrients as well as endogenous hormones interactions promoting the physiological changes and dormancy release. -
图 1 多花黄精种子生理后熟过程的表型分析
T0,后熟休眠期;T1,初始萌发期;T2,完全萌发期;T3,小球茎形成期;T4,胚芽形成期;T5,初生根茎形成期。
Figure 1. Superficial analysis on physiological post-ripening process of P. cyrtonema Hua seeds
T0: Post-mature dormancy; T1: initial germination stage; T2: complete germination stage; T3: corm formation stage; T4: germ formation stage; T5: primary rhizome formation stage.
图 3 多花黄精种子生理后熟过程的转录组测序分析
a: 差异表达基因数量; b: 主成分分析;c-e: T0vsT1, T0vsT2, T0vsT3, T0vsT4和T0vsT5五个比较组的共有、上调和下调差异基因韦恩图。
Figure 3. Transcriptome sequencing on physiological post-ripening process of P. cyrtonema Hua seeds
a: Number of differentially expressed genes; b: principal component analysis; c-e: shared, upregulated, and downregulated differential genes of 5 comparison groups (T0 vs. T1, T0 vs. T2, T0 vs. T3, T0 vs. T4, and T0 vs. T5), respectively.
表 1 荧光定量PCR引物序列
Table 1. Sequences of qPCR primers
基因名称
Gene name正向引物
Forward primer (5'-3')反向引物
Reverse prime (5'-3')TGA10 TGGTCGCCAATAATGCCTCC GAAGGCTTGCGCAGAATAGG IAA18 TTGCTGCTGGGAATCAGAGG ATGCGGTCTCCTTCATCGTC IAA30 TGCGGTACATCTTGAGGTCG GAGGAGTCCAGCACCAATCC TIFY 10 TTTCGGATTCGGGTAAGCCC CTGAATTCGGGTTCGGCTCT 内参基因 reference gene GCCTGTGGTTATCACTGCCT CGGATCGTCTGCTCCACTAC -
[1] 国家药典委员会. 中华人民共和国药典-一部: 2020年版[M]. 北京: 中国医药科技出版社, 2020. [2] 杨迎, 侯婷婷, 王威, 等. 黄精多糖的药理作用研究进展 [J]. 现代药物与临床, 2022, 37(3):659−665.YANG Y, HOU T T, WANG W, et al. Research progress on pharmacological effects of polysaccharides from Polygonati Rhizoma [J]. Drugs & Clinic, 2022, 37(3): 659−665. (in Chinese) [3] 李莉, 李钰, 杨绍杰. 基于网络药理学研究黄精丸防治阿尔茨海默病的作用机制 [J]. 中国医药科学, 2022, 12(13):76−81. doi: 10.3969/j.issn.2095-0616.2022.13.019LI L, LI Y, YANG S J. Investigation on the action mechanism of HJ pill in the prevention and treatment of Alzheimer’s disease based on network pharmacology [J]. China Medicine and Pharmacy, 2022, 12(13): 76−81. (in Chinese) doi: 10.3969/j.issn.2095-0616.2022.13.019 [4] CHEN X J, DUAN J F, LIU K Q, et al. Botany, traditional uses, and pharmacology of polygonati rhizoma [J]. Chinese Medicine and Culture, 2021, 4(4): 251−259. doi: 10.4103/CMAC.CMAC_39_21 [5] LI X L, ZHANG X X, MA R H, et al. Integrated miRNA and mRNA omics reveal dioscin suppresses migration and invasion via MEK/ERK and JNK signaling pathways in human endometrial carcinoma in vivo and in vitro [J]. Journal of Ethnopharmacology, 2023, 303: 116027. doi: 10.1016/j.jep.2022.116027 [6] 祝明珠, 俞年军, 史素影, 等. 多花黄精种子结构与休眠及萌发的关系研究 [J]. 种子, 2020, 39(3):7−12,19.ZHU M Z, YU N J, SHI S Y, et al. Study on the relationship between seed structure and germination of Polygonatum cyrtonema Hua [J]. Seed, 2020, 39(3): 7−12,19. (in Chinese) [7] 李询, 董诚明, 邢冰, 等. 多花黄精种子萌发抑制物特性研究 [J]. 种子, 2020, 39(6):108−110.LI X, DONG C M, XING B, et al. Study on the characteristics of seed germination inhibitors of Polygonatum cyrtonema Hua [J]. Seed, 2020, 39(6): 108−110. (in Chinese) [8] 王宏迪. 黄精种子胚乳细胞特异结构与种子休眠相关性研究[D]. 杨凌: 西北农林科技大学, 2018.WANG H D. The relative study of seeds dormancy and the specific structure of the endosperm cells of rhizoma polygonati seeds[D]. Yangling: Northwest A & F University, 2018. (in Chinese) [9] 陈怡, 柳雪晨, 陈松树, 等. 多花黄精种子萌发过程的形态和解剖研究 [J]. 种子, 2020, 39(2):5−10.CHEN Y, LIU X C, CHEN S S, et al. Morphological and anatomical studies during seed germination of Polygonatum cyrtonema Hua [J]. Seed, 2020, 39(2): 5−10. (in Chinese) [10] 安瑞朋. 中药材黄精种子萌发条件和休眠机制探究[D]. 保定: 河北农业大学, 2020.AN R P. Study on germination conditions and dormancy mechanism of Chinese medicinal material Polygonatum sibiricum seeds[D]. Baoding: Hebei Agricultural University, 2020. (in Chinese) [11] 刘佳, 朱翔, 王文祥, 等. 黄精种子休眠的研究进展 [J]. 农学学报, 2018, 8(3):11−15. doi: 10.11923/j.issn.2095-4050.cjas17100002LIU J, ZHU X, WANG W X, et al. Seeds dormancy of Polygonatum sibiricum red. : Research progress [J]. Journal of Agriculture, 2018, 8(3): 11−15. (in Chinese) doi: 10.11923/j.issn.2095-4050.cjas17100002 [12] 陈怡. 多花黄精种子和种茎萌发出苗的形态及生理研究[D]. 贵阳: 贵州大学, 2020.CHEN Y. Morphological and physiological study on germination and emergence of Polygonatum cyrtonema hua. seed and seed stem[D]. Guiyang: Guizhou University, 2020. (in Chinese) [13] 尉广飞, 李安宁, 徐佳, 等. 不同沙藏时间人参种子转录组分析及差异基因筛选 [J]. 药学学报, 2023, 58(10):3130−3139.WEI G F, LI A N, XU J, et al. Transcriptome analysis and differential gene screening of Panax ginseng seed in different sand storage stages [J]. Acta Pharmaceutica Sinica, 2023, 58(10): 3130−3139. (in Chinese) [14] PANG X A, SUO J T, LIU S, et al. Combined transcriptomic and metabolomic analysis reveals the potential mechanism of seed germination and young seedling growth in Tamarix hispida [J]. BMC Genomics, 2022, 23(1): 109. doi: 10.1186/s12864-022-08341-x [15] 高云鹏. 紫荆种子休眠解除过程中生理生化变化及分子机理研究[D]. 南京: 南京林业大学, 2020.GAO Y P. Study on the physiological and biochemical changes and molecular mechanisms in Cercis chinensis seeds during dormancy releasing[D]. Nanjing: Nanjing Forestry University, 2020. (in Chinese) [16] 刘保财, 陈菁瑛, 张武君, 等. 多花黄精种子萌发前后基因表达特征分析 [J]. 广西植物, 2023, 43(11):2078−2090. doi: 10.11931/guihaia.gxzw202210040LIU B C, CHEN J Y, ZHANG W J, et al. Analysis of gene expression characteristics before and after seed germination of Polygonatum cyrtonema [J]. Guihaia, 2023, 43(11): 2078−2090. (in Chinese) doi: 10.11931/guihaia.gxzw202210040 [17] 罗丽娜, 向增旭. 基于转录组测序分析的黄精种子休眠解除相关差异基因研究 [J]. 中国农学通报, 2021, 37(11):1−8. doi: 10.11924/j.issn.1000-6850.casb2020-0209LUO L N, XIANG Z X. The relative difference genes in the process of dormancy release of Polygonatum sibiricum Red. Based on the transcriptome sequencing analysis [J]. Chinese Agricultural Science Bulletin, 2021, 37(11): 1−8. (in Chinese) doi: 10.11924/j.issn.1000-6850.casb2020-0209 [18] 冷容, 胡彦婷, 周艳丽, 等. 人参种胚不同后熟发育阶段比较转录组学分析 [J]. 分子植物育种, 2020, 18(17):5638−5649.LENG R, HU Y T, ZHOU Y L, et al. Comparative transcriptome analysis of Panax ginseng C. A. mey. embryos in different after-ripening development stages [J]. Molecular Plant Breeding, 2020, 18(17): 5638−5649. (in Chinese) [19] 孙海燕, 李强, 朱铭玮, 等. 油用牡丹‘凤丹’种子层积过程中营养物质的代谢变化研究 [J]. 南京林业大学学报(自然科学版), 2021, 45(1):70−78.SUN H Y, LI Q, ZHU M W, et al. Dynamic changes of nutrients of Paeonia ostia‘Feng Dan’seed during its dormancy breaking [J]. Journal of Nanjing Forestry University (Natural Sciences Edition), 2021, 45(1): 70−78. (in Chinese) [20] 柳苗, 高捍东, 高燕, 等. 休眠解除过程中紫楠种子生理生化特征的变化 [J]. 南京林业大学学报(自然科学版), 2023, 47(2):9−17.LIU M, GAO H D, GAO Y, et al. Study on the physiological and biochemical changes of Phoebe sheareri seed during its dormancy breaking [J]. Journal of Nanjing Forestry University (Natural Sciences Edition), 2023, 47(2): 9−17. (in Chinese) [21] 文湘穗, 张誉荠, 王雨濛, 等. 外源激素处理对层积过程中北五味子种子生理生化的影响 [J]. 分子植物育种, 2023, 21(2):649−657.WEN X S, ZHANG Y Q, WANG Y M, et al. The effect of exogenous hormone treatment on Schisandra seed during stratification [J]. Molecular Plant Breeding, 2023, 21(2): 649−657. (in Chinese) [22] 赖佳辉, 赵艳丽, 蔡吹, 等. 重楼属植物种子休眠特性的研究进展[J/OL]. 分子植物育种. https://link.cnki.net/urlid/46.1068.S.20230911.1523.002LAI J H, ZHAO Y L, CAI C, et al. Research progress on seed dormancy characteristics of Paris L. [J/OL]. Molecular Plant Breeding. https://link.cnki.net/urlid/46.1068.S.20230911.1523.002(in Chinese) [23] 宋松泉, 唐翠芳, 雷华平, 等. 种子休眠与萌发调控的研究进展 [J]. 作物学报, 2024, 50(1):1−15.SONG S Q, TANG C F, LEI H P, et al. Research progress of seed dormancy and germination regulation [J]. Acta Agronmica Sinica, 2024, 50(1): 1−15. (in Chinese) [24] 久西加, 杜弢, 陈红刚, 等. 桃儿七种子低温层积过程中胚形态及生理生化物质变化 [J]. 西北植物学报, 2021, 41(12):2096−2103. doi: 10.7606/j.issn.1000-4025.2021.12.2096JIU X J, DU T, CHEN H G, et al. Changes of embryo morphology, physiology and biochemistry during low temperature stratification of Sinopodophyllum hexandrum seeds [J]. Acta Botanica Boreali-Occidentalia Sinica, 2021, 41(12): 2096−2103. (in Chinese) doi: 10.7606/j.issn.1000-4025.2021.12.2096 [25] 孙瑞敏, 王蕾, 王佳琪, 等. 裕民贝母破除种子生理休眠营养物质的变化 [J]. 分子植物育种, 2021, 19(15):5143−5149.SUN R M, WANG L, WANG J Q, et al. The changes of nutrients in breaking seed physiological dormancy of Fritillaria yuminensis X. Z. Duan [J]. Molecular Plant Breeding, 2021, 19(15): 5143−5149. (in Chinese) [26] 张武君, 赵云青, 刘保财, 等. 多花黄精种子层积过程生理变化研究 [J]. 福建农业学报, 2022, 37(8):995−1007.ZHANG W J, ZHAO Y Q, LIU B C, et al. Physiological changes of Polygonatum cyrtonema Hua seeds during stratification [J]. Fujian Journal of Agricultural Sciences, 2022, 37(8): 995−1007. (in Chinese) [27] 张春椿, 年慧慧, 李石清, 等. 激素加变温层积法处理南方红豆杉种子生理生化变化研究 [J]. 中华中医药杂志, 2012, 27(10):2723−2726.ZHANG C C, NIAN H H, LI S Q, et al. Study on physiological and biochemical changes of Taxus chinensis var. mairei seeds treated by hormone and variable temperature stratification [J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2012, 27(10): 2723−2726. (in Chinese) [28] 浦梅. 滇重楼种子发芽过程中生理生化特征研究[D]. 北京: 中国林业科学研究院, 2016PU M. Study on the physiological and biochemical characteristic during the seed germination of Paris polyphylla var. yunnanensis[D]. Beijing: Chinese Academy of Forestry, 2016. (in Chinese) -