图位克隆技术新型遗传学实验教学项目的设计与实践

doi:10.11983/CBB19107

摘要/Abstract

摘要： 染色体重组与连锁互换是遗传学教学的重点和难点。为使学生更好地理解此方面知识, 我们课题组前期利用图位克隆(map-based cloning)技术, 克隆了1个调控水稻(Oryza sativa)类病变表型的基因SPL5, 并基于此设计了一个新的综合型遗传学实验, 即利用DNA分子标记对基因进行定位。实验中学生利用水稻spl5突变体与野生型杂交获得的F₂代定位群体和多态性分子标记, 对spl5突变进行染色体连锁分析、初步定位和遗传作图。该教学实验不仅可有效促进学生对遗传学三大定律的理解, 而且对其开阔视野、提高解决问题和团队协作的能力也有促进作用。

关键词: 遗传学, 综合设计性实验, 图位克隆, 教学资源

Abstract: Recombination, linkage and crossover of chromosomes are the key and difficult points in genetics teaching. For students’ comprehensively understanding these concepts in teaching, we designed a new genetic experiment named ‘Gene mapping using DNA molecular markers’ based on the principle of the map-based cloning method, using our previous studies on cloning of a rice lesion-mimic phenotype regulating gene SPOTTED LEAF 5 (SPL5) as an example. In this experiment, students are instructed to perform the linkage analysis, genetically map the spl5 mutation and align a genetic map by using an F₂ population generated by a cross between spl5 and wild-type plants and polymorphic molecu- lar markers. This teaching experiment can not only effectively promote students' understanding of the three Laws of genetics, but also be helpful to broaden their vision and improve their ability to solve problems and work in a team.

Key words: genetics, comprehensive designing experiment, map-based cloning, educational resources

陈析丰,刘亚萍,马伯军. 图位克隆技术新型遗传学实验教学项目的设计与实践. 植物学报, 2019, 54(6): 797-803.

Xifeng Chen,Yaping Liu,Bojun Ma. Design and Practice of a New Teaching Project of the Map-based Cloning Experiment in Genetics. Chinese Bulletin of Botany, 2019, 54(6): 797-803.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19107

https://www.chinbullbotany.com/CN/Y2019/V54/I6/797

图/表 5

图1 减数分裂过程中目标基因与分子标记的重组方式 A与a代表1对显性和隐性等位基因, A对于a为显性, AA、Aa为显性表型, aa为隐性表型; M与m代表染色体上位于同一位点的任一分子标记, M来自显性亲本(AA), m来自隐性亲本(aa)。

Figure 1 Recombination patterns between the target gene and the molecular markers during meiosis A and a represent a pair of dominant and recessive alleles, of which AA and Aa show dominant phenotypes and aa shows a recessive phenotype; M and m represent a given molecular marker at the same locus derived from the dominant parent (AA) and the recessive parent (aa), respectively.

图2 水稻类病变spl5突变体的表型与基因定位群体的构建 (A) 对照粳稻日本晴及spl5突变体植株表型; (B) 日本晴及spl5突变体叶片表型; (C) 野生型籼稻品种9311与粳稻日本晴背景的spl5突变体杂交构建F2代定位群体流程。

Figure 2 The phenotype of the rice spl5 lesion mimic mutant and a schematic workflow of generation of the genetic mapping population (A) The plant phenotype of wild-type control Nipponbare, a Japonica rice cultivar, and the spl5 mutant; (B) The leaf phenotype of wild-type control and the spl5 mutant; (C) A workflow showing the generation of an F2 mapping population by crossing an Indica cultivar 9311 with the spl5 mutant.

表1 本研究SSR标记的PCR引物

Table 1 PCR primers of the SSR markers used in this study

SSR标记	正向引物 (5°-3°)	反向引物 (5°-3°)
RM21103	ACAAGTCCACAAGGACCACAACC	TGCTCCACCCAAAGATACAGAGC
RM21309	GAGTGGATTTACTGACAAGCTCTTCC	GATTCTCTTGGAGCCCATACTCC
RM21470	TCTTGCCATCACATAGCAACAGG	ACTCGGTGAGCATCCAATGTCC
RM22426	ATTTCGTCGTCGTGTCTGTCC	TTAAAGCAGCCTGATCGAAAGC
RM22648	AGCAGCTCCGATTGTGTTAGCC	AATGGCGTCTGTGGTACTTTGC
RM22925	CGATTACCAGGACAACAGATCACG	GTGGCTCTTGAGTGTACGTGTGC

图3 SPL5基因的连锁分析与遗传定位 (A) 本实验SSR标记所在的染色体及其物理距离; (B) 本实验SSR标记PCR扩增条带的电泳图; (C) SPL5基因的染色体遗传连锁图。

Figure 3 Linkage analysis and genetic mapping of the SPL5 gene (A) A physical map of the SSR markers on chromosomes used in this study; (B) Analysis of the PCR products of the SSR markers by electrophoresis; (C) A genetic linkage map of the SPL5 gene.

表2 本研究SSR标记基因型分离比的卡方(χ2)检验

Table 2 The Chi-square (χ2) test for the genotype separation ratio of the SSR markers in this study

分子标记	20个F₂突变型单株中各基因型的数量			χ²值	P值
分子标记	MM	Mm	mm	χ²值	P值
RM21103	0	0	20	60	<0.01
RM21309	0	3	17	38.7	<0.01
RM21470	1	7	12	13.9	<0.01
RM22426	4	11	5	0.3	>0.05
RM22648	5	9	6	0.3	>0.05
RM22925	7	10	3	1.6	>0.05

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