Single-microspore Microdissection of Hevea brasiliensis

doi:10.3969/j.issn.1000-2561.2020.08.003

Abstract

Abstract:

Hevea brasiliensis is an important tropical crop. In this study, the young male flowers of ‘Reyan 7-33-97’ in the meiotic tetrad period were used as the experimental materials to obtain single cells. Four enzymatic solutions, 5% cellulose, 5% pectinase, 5% β-glucanase, 5% cellulase and 4% pectinase mixed enzyme solution, were used in the experiment to explore the best scheme for the degradation of tetrad callose. As a result, under the condition of 1 h and 37℃, 5% cellulase and 4% pectinase mixed enzyme solution had the best enzymatic hydrolysis effect, and the single microspore with good quality could be obtained. Through the continuous exploration and experiment on the fine length of the needle tip, the diameter of the sharpening of the needle and the micro-operation separation system, a set of microdissection technology system suitable for the single microspore of ‘Reyan 7-33-97’ was successfully established. Single-microspores were the direct products of meiosis. The separation and analysis of single cells could accurately resolve gene recombination and exchange during the meiosis of rubber trees, and determine the genomic variation. The study would provide theoretical and technical support for single-cell whole genome sequencing of H. brasiliensis.

Key words: Hevea brasiliensis, microdissection, single cell, tetrad

CLC Number:

Q943

YANG Xingxing,WANG Ying,ZHUANG Nansheng,GAO Heqiong,WANG Yuxiang. Single-microspore Microdissection of Hevea brasiliensis[J]. Chinese Journal of Tropical Crops, 2020, 41(8): 1522-1528.

Figures/Tables 7

Tab. 1 Factors of enzymatic hydrolysis of tetrad callose of Hevea brasiliensis ‘Reyan 7-33-97’

水平 Level	因素Factor
水平 Level	酶Enzyme	温度Temperature/℃	时间Time/h
1	5%纤维素酶	37	1
2	5%果胶酶	50	3
3	5% β-葡聚糖酶		6
4	5%纤维素酶与4%果胶酶混合酶

Tab. 2 Size of male flowers during male gametogenesis H. brasiliensis ‘Reyan 7-33-97’

花长度 Length of flower/mm	花直径 Diameter of flower/mm	雄配子发育时期 Period of male gamete development
1.7~1.8	0.6~0.7	小孢子母细胞早期
1.8~1.9	0.7~0.9	小孢子母细胞中后期
1.9~2.1	0.9~1.2	四分体时期
2.2~2.4	1.2~1.4	单核小孢子时期
2.5~3.0	1.4~1.7	花粉成熟期

Fig. 1 Male flower in tetrad period of H. brasiliensis ‘Reyan 7-33-97’

Fig. 2 Microscopic separation process of single tetrad of H. brasiliensis ‘Reyan 7-33-97’ A: Tetrad in the liquid; B: Separation of single tetrad; C: Separated liquid phase of single tetrad.

Tab. 3 Enzymatic efficiency of tetrad callose under different enzyme types and enzymatic hydrolysis time

酶 Enzyme	时间 Time/h	37℃下酶解率 Enzymolysis rate at 37℃/%	50℃下酶解率 Enzymolysis rate at 50℃/%
5%纤维素酶	1	75	65
	3	90	88
	6	98	95
5%果胶酶	1	0	0
	3	25	32
	6	80	85
5% β-葡聚糖酶	1	0	0
	3	0	0
	6	20	25
5%纤维素酶与4%果胶酶混合酶	1	100	100
	3	100	100
	6	100	100

Fig. 3 Degradation of tetrad callose of H. brasiliensis ‘Reyan 7-33-97’ by four enzymes A: 5% cellulase and 4% pectinase mixed enzyme solution for 1 h; B: 5% cellulase for 1 h; C: 5% pectinase for 1 h; D: 5% β-glucanase for 1 h; E: 5% cellulase for 3 h; F: 5% pectinase for 3 h; G: 5% β-glucanase for 3 h; H: 5% cellulase for 6 h; I: 5% pectinase for 6 h; J: 5% β-glucanase for 6 h.

Fig. 4 Microscopic separation process of single single microspore of H. brasiliensis ‘Reyan-7-33-97’ A: Single tetrad in the liquid; B: Four single cells of the same tetrad in the liquid; C-F: Separation of four single cells of the same tetrad one by one.

References 37

[1]	陈学俊. 巴西橡胶树热研7-33-97第1, 5和9号染色体的分离及文库的构建[D]. 海口: 海南大学, 2014.
[2]	何宇清, 孙蒙祥. 单细胞技术进展及其在植物研究中的应用[J]. 植物科学学报, 2016,34(3):475-487. DOI URL
[3]	Wright G, Tucker M J, Morton P C, et al. Micromanipulation in assisted reproduction: a review of current technology[J]. Current Opinion in Obstetrics & Gynecology, 1998,10(3):221-226. URL PMID
[4]	毕春明, 陈大元. 哺乳动物胚胎分割研究进展及应用前景[J]. 生理科学进展, 2002,33(3):205-208.
[5]	李志, 汪洋, 周莉, 等. 银鲫卵母细胞体外诱导成熟技术的建立[J]. 水生生物学报, 2017,41(5):984-991.
[6]	杨文涛, 许良中. 从组织切片上分离单个细胞的相关技术及其应用前景[J]. 诊断病理学杂志, 2001(2):124-125.
[7]	Li C X, Wang G Q, Li W S, et al. New cell separation technique for the isolation and analysis of cells from biological mixtures in forensic caseworks[J]. Croatian Medical Journal, 2011,52(3):293-298. DOI URL PMID
[8]	Brehm-Stecher B F, Johnson E A. Single-cell microbiology: tools, technologies, and applications[J]. Microbiology and Molecular Biology Reviews, 2004,68(3):538-559. DOI URL PMID
[9]	Sherman F. Micromanipulator for yeast genetic studies[J]. Applied Microbiology, 1973,26(5):829. URL PMID
[10]	Frohlich J, Konig H. New techniques for isolation of single prokaryotic cells[J]. FEMS Microbiology Reviews, 2000,24(5):567-572. DOI URL PMID
[11]	陈学俊, 王英, 高和琼, 等. 巴西橡胶树‘热研7-33-97’单染色体显微分离与扩增[J]. 中国农学通报, 2014,30(13):29-33.
[12]	彭永康, 于玲, 霍昕, 等. 用显微分离和超敏感银染电泳方法研究百合减数第一分裂周期蛋白质变化[J]. 植物研究, 2001,21(2):313-316.
[13]	于建春, 金绍波, 赵建, 等. 一种分析有丝分裂期蛋白的新方法[J]. 天津师范大学学报(自然科学版), 2001,21(3):65-66, 70.
[14]	Trouillon R, Passarelli M K, Wang J, et al. Chemical analysis of single cells[J]. Analytical Chemistry, 2013,85(2):522-542. DOI URL PMID
[15]	Wang J, Fan H, Behr B, et al. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm[J]. Cell, 2012,150(2):402-412. DOI URL PMID
[16]	Hou Y, Fan W, Yan L, et al. Genome analyses of single human oocytes[J]. Cell, 2013,155(7):1492-1506. URL PMID
[17]	Ren S C, Qu M, Sun Y H. Investigating intratumour heterogeneity by single-cell sequencing[J]. Asian Journal of Andrology, 2013,15(6):729-734. URL PMID
[18]	Shirai M, Taniguchi T, Kambara H. Emerging applications of single-cell diagnostics[J]. Topics in Current Chemistry, 2014,336:99-116. DOI URL PMID
[19]	Rosenfeld N. Gene regulation at the single-cell level[J]. Science, 2005,307(5717):1962-1965. URL PMID
[20]	李响. 利用单细胞测序技术剖析玉米遗传重组交换和单倍体诱导的机制[D]. 武汉: 华中农业大学, 2017.
[21]	Kubo M, Nishiyama T, Tamada Y, et al. Single-cell transcriptome analysis of physcomitrella leaf cells during reprogramming using microcapillary manipulation[J]. Nucleic acids Research, 2019,47(9):4539-4553. DOI URL PMID
[22]	冯素萍, 李维国, 于飞, 等. 巴西橡胶树SSR遗传图谱的构建[J]. 遗传, 2010,32(8):857-863.
[23]	刘正林, 庄南生, 王英, 等. 巴西橡胶树HbWRKY基因家族10个成员在染色体上的定位[J]. 植物遗传资源学报, 2018,19(6):1170-1179.
[24]	Tang C R, Yang M, Fang Y J, et al. The rubber tree genome reveals new insights into rubber production and species adaptation[J]. Nature Plants, 2016,2(6):16073. DOI URL PMID
[25]	孙爱花, 李哲, 黄天带. 橡胶树花药的培养[J]. 植物生理通讯, 2006,42(4):785-789.
[26]	冯斗, 王建岭, 席世丽, 等. 木薯根尖染色体的观察技术[J]. 安徽农业科学, 2008,36(9):3711-3712.
[27]	高和琼, 王英, 金鸽, 等. 橡胶树叶片染色体制片方法的优化[J]. 热带作物学报, 2009,30(5):565-569.
[28]	陈学俊, 王英, 高和琼, 等. 巴西橡胶树‘热研7-33-97’单染色体显微分离与扩增[J]. 中国农学通报, 2014,30(13):29-33.
[29]	张源源, 李国华, 龙青姨, 等. 橡胶树不同品种雌花分布和座果规律研究[J]. 植物科学学报, 2013,31(3):242-247.
[30]	刘晓瑞, 陈祖铿, 刘家熙. 植物小孢子母细胞减数分裂过程中胼胝质染色的新方法[J]. 植物学通报, 2008,25(4):455-458.
[31]	王元军. 植物体内的胼胝质[J]. 生物学通报, 2005(1):18-19.
[32]	郝建华, 强胜. 被子植物有性和无融合生殖过程中胼胝质壁的动态变化及其生物学意义[J]. 植物生理通讯, 2006,42(1):141-147.
[33]	Chen X Y, Kim J Y. Callose synjournal in higher plants[J]. Plant Signal Behavior, 2009,4(6):489-492.
[34]	谢石文, 邱德勃, 王哲魁, 等. 橡胶树花药和小孢子发生与发育过程的观察[J]. 华南植物学报, 1993(1):25-30.
[35]	邓秀新, 章文才. 柑桔四分体小孢子原生质体的分离[J]. 中国柑桔, 1990(1):7-8.
[36]	王发明, 王平, 唐琦, 等. 柚(Citrus grandis Osbeck)单染色体LA-PCR-AFLP技术体系中外源污染的防控[J]. 热带作物学报, 2010,31(12):2135-2141.
[37]	殷卉, 王英, 高和琼, 等. 巴西橡胶树单染色体显微分离及其两种体外扩增方法比较[J]. 分子植物育种, 2015,13(11):2584-2589.