响应面法优化余甘子种质资源ISSR反应体系

王建超; 何银莺; 黄旭萍; 沈朝贵; 陈发兴; 郭林榕

doi:10.19303/j.issn.1008-0384.2021.07.004

响应面法优化余甘子种质资源ISSR反应体系

doi: 10.19303/j.issn.1008-0384.2021.07.004

王建超^{1, 2,},
何银莺²,
黄旭萍²,
沈朝贵¹,
陈发兴²,
郭林榕^1, ,

1.
福建省农业科学院果树研究所，福建　福州　350013
2.
福建农林大学园艺学院，福建　福州　350002

基金项目: 福建省科技计划公益类专项（2019R1028-11）；国家热带植物种质资源库（余甘子种质资源分库）（NTPGRC2021-011）；农业农村部物种品种（热带作物）资源保护项目（151821301354052701）

详细信息

作者简介:
王建超（1988−），男，研究实习员，研究方向：热带与亚热带果树种质资源保护与生理生化研究（Email：447327289@qq.com）

通讯作者:
郭林榕（1963−），女，副研究员，研究方向：热带与亚热带果树种质资源保存、选育种及栽培研究（Email：linrong-g@163.com）

中图分类号: S 682.310.36
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出版历程
- 收稿日期: 2021-03-16
- 修回日期: 2021-05-12
- 刊出日期: 2021-07-28

Response Surface Optimization of ISSR-PCR Reaction for Genetic Study on Phyllanthus Emblica

1.
Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的优化余甘子种质资源ISSR-PCR反应体系，为余甘子种质资源遗传多样性及亲缘关系研究提供基础。方法以缅甸、印度、广东、云南、福建等5份来源不同的余甘子种质资源的基因组DNA组成混合的DNA模板，综合单因素试验和响应面分析法，分析引物浓度、2×Taq Master Mix添加量、DNA模板量、退火温度等反应条件对ISSR-PCR反应体系的影响，优化建立余甘子ISSR-PCR反应体系。结果引物浓度和2×Taq Master Mix添加量对扩增效果有较大影响，DNA模板量影响较小；引物浓度和DNA模板量交互作用明显；余甘子ISSR反应体系为引物浓度0.4 μmol·L⁻¹，2×Taq Master Mix添加量13 μL，DNA模板量30 ng，扩增结果与响应面分析模型理论值相对误差仅为9.39%；退火温度为50.5～52.7 ℃时，随着温度的升高，条带质量变好，数量变多，退火温度为52.7 ℃时可获得多样性好的清晰条带。结论获得ISSR-PCR反应体系为引物浓度0.4 μmol·L⁻¹，2×Taq Master Mix添加量13 μL，DNA模板量30 ng，退火温度52.7 ℃，扩增循环数35循环，扩增获得的条带清晰、稳定，多样性好，该体系适于余甘子种质资源的遗传多样性和亲缘关系等分析研究。
- 余甘子 /
- ISSR-PCR /
- 响应面 /
- 体系优化
Abstract: Objective ISSR-PCR reaction system for genetic study on Phyllanthus emblica germplasms was optimized. Methods A mixed DNA template composed of genomic DNA of P. emblica germplasms came from Myanmar, India, Guangdong, Yunnan, and Fujian was obtained. The single factor test and response surface analysis were used to optimize the ISSR-PCR reaction conditions including primer concentration, amount of 2×Taq Master Mix, DNA template amount, and annealing temperature. Result The primer concentration and addition amount of 2×Taq Master Mix had a greater impact on the amplification than did the DNA template amount. A significant interaction between the primer concentration and DNA template amount was found. The optimized system with a primer concentration of 0.4 μmol·L⁻¹, a 2×Taq Master Mix of 13 μL, and a DNA template concentration of 30 ng was established that achieved a low relative error of 9.39% in predicting the theoretical response. Within the annealing temperature between 50.5 ℃ and 52.7 ℃, increasing temperature improved the number and quality of bands. When the annealing temperature is 50.5-52.7 ℃, the number of strips increases, and the quality of the strips becomes better with the increase of temperature. The annealing temperature is 52.7 ℃ to obtain good diversity and clear bands. Conclusion The optimized ISSR-PCR reaction system was established applying a primer concentration of 0.4 μmol·L⁻¹, a 2×Taq Master Mix of 13 L, a DNA template concentration of 30 ng at the annealing temperature of 52.7 ℃ for 35 amplification cycles. The methodology could be used to study the genetic diversity and relationship of P. emblica germplasms.
- Phyllanthus emblica /
- ISSR-PCR /
- response surface methodology /
- system optimization

HTML全文

图 1 余甘子基因组DNA提取电泳效果

注：M为DL15K DNA marker；1.缅甸实生；2.印度大果；3.甜种；4.盈玉；5.福建本地种。

Figure 1. Electrophoretic DNA extraction on germplasms

Note: M: DL 15K DNA marker; 1: Myanmar seedling; 2: a wild variety from India; 3: Tianzhong; 4: Yingyu; 5:Fujian native species.

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图 2 引物浓度（UBC 841）、2×Taq Master Mix添加量、DNA模板量对ISSR反应体系的影响

注：M为DL15K DNA Maker；A、B、C分别表示引物浓度、2×Taq Master Mix添加量、DNA模板量单因素试验，1～15代表不同处理。

Figure 2. Effect of primer concentration, 2×Taq Master Mix addition, and DNA template amount on ISSR reaction

Note: M: DL15K DNA maker; A, B, and C: primer concentration, 2×Taq Master Mix addition, and DNA template amount, respectively, in single factor test; and 1-15: various treatments.

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图 3 17组响应面试验电泳图谱

注：M为DL 2K DNA Maker；1～17分别对应表4序号ISSR反应体系电泳谱图。

Figure 3. Seventeen sets of electropherograms by response surface test

Note: M: DL 2K DNA maker; 1-17: electrophoresis spectra of ISSR reaction system shown on Table 4.

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图 4 引物浓度（UBC841）和DNA模板量对电泳图谱评分的影响

Figure 4. Effects of primer UBC841 concentration and DNA template amount on electrophoretic scores

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图 5 验证试验电泳图谱

注：M为DL2K DNA Marker；A、B与C、D分别为验证试验1、2电泳结果。

Figure 5. Electropherogram of validation test

Note: M: DL15K DNA marker; A and B: results of validation test 1; C and D: results of validation test 2.

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图 6 退火温度对ISSR反应体系的影响

注：M为DL2K DNA Marker；电泳谱1～8依次表示50.5 、50.7、51.2 、51.9 、52.7 、53.3、53.7 和54.0 ℃退火温度下扩增效果。

Figure 6. Effect of annealing temperature on ISSR assay

Note: M: DL2K DNA marker; Electrophoresis 1-8: at ISSR annealing temperatures of 50.5 , 50.7 , 51.2 , 51.9, 52.7, 53.3 , 53.7 and 54.0 ℃, respectively.

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图 7 引物UBC841对不同余甘子种质资源扩增的电泳结果

注：M.DL 2K DNA Marker；1～5表示不同来源的余甘子种质资源。

Figure 7. Electrophoretic results on amplification of germplasms using primer UBC841

Note: M: DL 2K DNA marker; 1-5: 5 P. emblica germplasms.

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表 1 试验材料来源

Table 1. Sources of testing materials

编号 Code	品种（系）名　　 Name	来源 Source	采样地 Sample Site
1	缅甸实生 Myanmar seedling	缅甸 Myanmar	余甘子种质资源圃 Field genebank for Phyllanthus emblica
2	印度大果 India species	印度 India	余甘子种质资源圃 Field genebank for Phyllanthus emblica
3	甜种 Tianzhong	中国广东 Guangdong, China	余甘子种质资源圃 Field genebank for Phyllanthus emblica
4	盈玉 Yingyu	中国云南 Yunnan, China	余甘子种质资源圃 Field genebank for Phyllanthus emblica
5	福建本地种 Fujian native specie	中国福建 Fujian, China	余甘子种质资源圃 Field genebank for Phyllanthus emblica

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表 2 单因素试验设计

Table 2. Simple factor experiment

序号 Number	编号 Code	引物浓度 Primer concentration/ （μmol·L⁻¹）	2×Taq Master Mix 添加量 2×Taq Master Mix addition amount/μL	DNA模板量 DNA template amount/ng
1	A	0.2	12	45
2		0.3	12	45
3		0.4	12	45
4		0.5	12	45
5		0.6	12	45
6	B	0.4	8	45
7		0.4	10	45
8		0.4	12	45
9		0.4	14	45
10		0.4	16	45
11	C	0.4	12	15
12		0.4	12	30
13		0.4	12	45
14		0.4	12	60
15		0.4	12	75
注：在整个反应过程中，随着比较因素梯度的设置变动，相应的调整ddH₂O的量以保证反应体系为25 μL。 Note: During reaction process, amount of added ddH₂O was continually adjusted to maintain volume of reaction system at 25 μL as gradient of comparison factor changed.

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表 3 响应面分析因子及水平表

Table 3. Factors and levels of response surface design

反应条件 Reation Condition	编码 Code	水平 Levels
反应条件 Reation Condition	编码 Code	−1	0	1
引物浓度 Primer concentration/（μmol·L⁻¹）	X₁	0.30	0.35	0.40
2×Taq Master Mix 添加量 2×Taq Master Mix addition amount/μL	X₂	12	13	14
DNA模板量 DNA template amount/ng	X₃	25	30	35

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表 4 响应面分析方案及试验结果

Table 4. Design and results on factors and levels of response surface test

序号 Order number	X₁	X₂	X₃	评分 Score
1	0	0	0	14.2±1.41
2	0	1	−1	4.25±1.26
3	1	0	1	2.12±1.26
4	1	−1	0	9.25±0.5
5	−1	−1	0	10.25±0.82
6	1	0	−1	6.75±0.96
7	0	−1	1	6.38±1.60
8	1	1	0	9.38±0.95
9	0	−1	−1	8.13±0.85
10	0	0	0	13.55±2.46
11	0	1	1	7.38±1.38
12	0	0	0	10.00±1.83
13	1	0	1	5.13±0.82
14	0	1	0	8.75±0.96
15	−1	0	−1	2.25±0.50
16	−1	0	1	7.50±1.29
17	0	0	0	14.75±5.19

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表 5 验证试验与结果

Table 5. Validation test results

序号 Number	理论解决方案 Theoretical solution	实际解决方案 Practical solution	理论评分 Theoretical score	实际评分 Actual score	相对误差 Relative error/%
1	X₁：0.36 μmol·L⁻¹，X₂：13.46 μL，X₃：29.41 ng	X₁：0.35 μmol·L⁻¹，X₂：13.5 μL，X₃：30 ng	13.1149	11.4±0.55	9.58
2	X₁：0.37 μmol·L⁻¹，X₂：13.09 μL，X₃：29.06 ng	X₁：0.40 μmol·L⁻¹，X₂：13.0 μL，X₃：30 ng	13.1304	12.5±0.79	9.39

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