番茄匍柄霉原生质体的制备与再生体系构建

doi:10.3969/j.issn.1004-1524.2022.02.15

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (2): 337-342.DOI: 10.3969/j.issn.1004-1524.2022.02.15

番茄匍柄霉原生质体的制备与再生体系构建

龚伟杰(), 刘洪, 黄萍, 王慧, 周倩^*()

湖南农业大学植物保护学院,植物病虫害生物学与防控湖南省重点实验室,湖南长沙 410128

收稿日期:2021-05-24 出版日期:2022-02-25 发布日期:2022-03-02
通讯作者: 周倩
作者简介:周倩,E-mail: zhouqian2617@hunau.edu.cn
龚伟杰(1997—),男,湖南常德人,硕士研究生,研究方向为分子植物病理学。E-mail: 1374489069@qq.com
基金资助:
湖南省教育厅创新平台项目(19K040);湖南省自然科学基金(2018JJ3220)

Optimization of protoplast preparation and regeneration conditions of Stemphylium lycopersici

GONG Weijie(), LIU Hong, HUANG Ping, WANG Hui, ZHOU Qian^*()

Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insects Pests, College of Plant Protection, Hunan Agricultural University, Changsha 410128, China

Received:2021-05-24 Online:2022-02-25 Published:2022-03-02
Contact: ZHOU Qian

摘要/Abstract

摘要：

以番茄匍柄霉(Stemphylium lycopersici)CS12菌株为研究对象,从菌龄、酶种类与浓度、酶组合、酶解温度与时间、不同渗透压稳定剂等方面对番茄匍柄霉原生质体制备条件进行了优化,并筛选了最适再生培养基,成功建立番茄匍柄霉原生质体制备与再生体系。番茄匍柄霉CS12在菌龄为36 h,稳渗剂为0.7 mol·L^-1 NaCl溶液,酶解液为10 mg·mL^-1Kitalase裂解酶,28 ℃酶解3.5 h的条件下制备的原生质体效果好,在YEPS再生培养基上再生效率最高。番茄匍柄霉原生质体制备与再生体系的建立,为进一步研究番茄匍柄霉致病机理与功能基因组学奠定了基础。

关键词: 番茄匍柄霉, 原生质体, 再生, 稳渗剂, 菌龄

Abstract:

Protoplastpreparation conditions ofStemphylium lycopersiciwas optimizedusing strain CS 12 fromhypha age, enzyme species and concentration, enzyme combination, enzymolysis temperature and time, isotonic solutions, and the optimal regeneration medium. Theprotoplasm preparation and regeneration of S.lycopersici was successfully established by using mycelium cultured 36 h, 0.7 mol·L^-1 NaCl solution as the osmotic stabilizer, 10 mg·mL^-1Kitalase as enzymatic hydrolysate at 28℃ for 3.5 h, and the regeneration efficiency was the highest on the YEPS regeneration medium. The establishment of the protoplast preparation and regeneration of S.lycopersici laid foundation for further research on functional genomics and pathogenic mechanism of S.lycopersici.

Key words: Stemphylium lycopersici, protoplast, regeneration, osmotic stabilizer, hypha age

中图分类号:

S432.4⁺4

龚伟杰, 刘洪, 黄萍, 王慧, 周倩. 番茄匍柄霉原生质体的制备与再生体系构建[J]. 浙江农业学报, 2022, 34(2): 337-342.

GONG Weijie, LIU Hong, HUANG Ping, WANG Hui, ZHOU Qian. Optimization of protoplast preparation and regeneration conditions of Stemphylium lycopersici[J]. Acta Agriculturae Zhejiangensis, 2022, 34(2): 337-342.

图/表 8

图1 稳渗剂对原生质体制备的影响

Fig.1 Effect of osmotic stabilizers on preparation of protoplasts

图2 酶种类与浓度对原生质体制备的影响

Fig.2 Effects of different enzyme types and concentrations on protoplasts preparation

图3 不同酶组合对原生质体制备的影响 Kit,Kitalase裂解酶;Lys,溶壁酶;Dri,崩溃酶;Sna,蜗牛酶。

Fig.3 Effect of different enzyme combinations on protoplasts preparation Kit, Kitalase; Lys, Lysing enzymes; Dri, Driselase; Sna, Snailase.

图4 不同菌龄对原生质体制备的影响

Fig.4 Effect of growth time of fungi on preparation of protoplasts

图5 不同酶解温度对原生质体制备的影响

Fig.5 Effect of enzymolysis temperature on preparation of protoplasts

图6 不同酶解时间对原生质体制备的影响

Fig.6 Effect of enzymolysis time on preparation of protoplasts

图7 番茄匍柄霉CS12的原生质体形态

Fig.7 Protoplast of Stemphylium lycopersici CS12

图8 不同培养基上的原生质体再生率

Fig.8 Regeneration rate of protoplast in different medium

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番茄匍柄霉原生质体的制备与再生体系构建

Optimization of protoplast preparation and regeneration conditions of Stemphylium lycopersici

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