《植物生理学报》 2016, 52(3): 259-268

低温诱导下苹果花药差异表达基因分析

罗慧珍^1,2, 邓舒³, 张春芬³, 肖蓉³, 王卉⁴, 孟玉平², 曹秋芬^1,2,*

¹山西大学生物工程学院, 太原030006; ²山西省农业科学院生物技术研究中心, 太原030031; ³山西省农业科学院果树研究所,山西太谷030815; ⁴山西省农业科学院科研处, 太原030031

通信作者：曹秋芬；E-mail: qiufengcao@163.com

摘　要：

低温处理是苹果花药培养诱导胚状体形成的关键步骤, 花药中的小孢子在经过一定时间的低温诱导后才能获得胚性潜能。本文通过转录组测序的方法对低温处理前和低温处理30 d的苹果花药进行研究, 分析低温诱导条件下花药中的差异表达基因。结果表明: 转录组测序共得到10.90 Gb的Clean Data。基因表达分析结果显示, 共有4 105个基因发生差异表达, 包括表达上调基因1 849个, 表达下调基因2 256个。注释到GO、COG、KEGG、Swiss-Prot和nr数据库的差异表达基因分别有3 325个、1 504个、733个、2 993个和3 758个。差异表达基因主要富集在与糖类代谢和激素信号转导有关的过程中, 其中在淀粉和蔗糖代谢、植物激素信号转导这两个代谢通路中富集的差异表达基因最多。筛选出的差异表达基因中控制蔗糖合成、细胞分裂素、脱落酸和油菜素内酯信号转导的相关基因表达量上调, 控制淀粉合成、生长素信号转导的相关基因表达量下调。差异表达基因的荧光定量PCR结果显示测序结果和实际结果变化趋势完全一致。由此可见, 苹果花药经低温诱导后, 影响蔗糖、淀粉生物合成和生长素、细胞分裂素、脱落酸、赤霉素和油菜素内酯信号转导相关基因的表达变化是影响小孢子获得胚性潜能的关键。

关键词：苹果花药; 胚状体; 转录组测序; 差异表达基因; 荧光定量PCR

收稿：2015-10-21   修定：2016-01-28

资助：国家自然基金(31372033)、国家科技基础性工作专项(2012, FY110100-5)和山西省青年科技研究基金(2015021153)。

Differentially expressed gene analysis of apple (Malus domestica) anther under low temperature induction

LUO Hui-Zhen^1,2, DENG Shu³, ZHANG Chun-Fen³, XIAO Rong³, WANG Hui⁴, MENG Yu-Ping², CAO Qiu-Fen^1,2,*

¹College of Biological Engineering, Shanxi University, Taiyuan 030006, China; ²Biotechnology Research Center, Shanxi Academy of Agriculture Sciences, Taiyuan 030031, China; ³Fruit Tree Research Institute, Shanxi Academy of Agriculture Sciences, Taigu, Shanxi 030815, China; ⁴Research Department, Shanxi Academy of Agriculture Sciences, Taiyuan 030031, China

Corresponding author: CAO Qiu-Fen; E-mail: qiufengcao@163.com

Abstract:

Low temperature treatment is the key step in the induction of the formation of embryoid during apple anther culture, and pollen in anthers can acquire the embryogenic potential after a certain period of low temperature induction. In this study, the research on the two group apple anther that one was treated by 30 days low temperature and the other untreated were carried out by the method of RNA-Seq, and the DEG (differentially expressed genes) in the process of embryoid formation was analyzed. The results showed that: A total of 10.90 Gb clean data was generated using the Illumina HiSeq 2 500 system. Gene expression revealed 1 849 up-regulated genes and 2 256 down-regulated genes among the 4 105 DEG. By blasting to different databases, 3 325 DEG was annotated by Go terms; 1 504 DEG was able to be assigned to COG database; 733 DEG was annotated with KEGG blast; 2 993 and 3 758 DEG were annotated by Swiss-Prot and nr databases, respectively. DEG was mainly enriched in the metabolic processes associated with carbohydrate metabolism and hormone signal transduction, and DEG in the pathway starch and sucrose metabolism and plant hormone signal transduction were the most. DEG which control sucrose biosynthesis, cytokinin, abscisic acid and brassinosteroid’ s signal transduction were up-regulated while those control starch biosynthesis and auxin’ s signal transduction were down-regulated. Besides, the RT-qPCR results showed the sequencing results were consistent with the actual results. From here we see that, changes of the genes that affecting sucrose, starch biosynthesis, auxin, cytokinin, abscisic acid, gibberellin and brassinosteroid’ s signal transduction were key to the process of apple anther embryoid formation induced by low temperature.

Key words: apple anther; embryoid; RNA-Seq; differentially expressed genes; RT-qPCR