马尾松球果发育不同时期qRT-PCR内参基因的筛选与评价毕业论文_林学毕业论文

马尾松球果发育不同时期qRT-PCR内参基因的筛选与评价毕业论文

2021-04-19更新

摘 要

马尾松(Pinus massoniana Lamb.)与黄山松(Pinus hwangshanensis Hsia.)主要分布于我国南方地区,前者是重要的造林树种和经济林树种,后者为高海拔地区观赏树种。在长江中下游地区,马尾松主要生长在海拔700m以下地区,而黄山松则多数分布于海拔900m以上地带,在海拔700-900m地区存在他们的渐渗杂种,本文中称之为杂种松。本研究以不同发育时期的马尾松和杂种松球果为实验材料,根据内参基因的文献报道,通过将常用的内参基因序列与转录组数据中unigene序列进行比对确定了actin(ACT)、protein TOC75-3(TOC75)、ubiquitin(UBQ)、phosphoglycerate kinase 1(PGK1)、succinate dehydrogenase(SDH)、dystrophia myotonica WD repeat-containing protein(DMWD)、uncharacterized protein LOC103705956(upLOC)和F-box family protein(FBOX)共8个候选内参基因,采用实时荧光定量PCR技术并利用3种软件(geNorm、NormFinder和BestKeeper)对候选内参基因的表达稳定性进行了评价。结果表明,upLOC和FBOX在马尾松和杂种松球果发育不同时期中均表达稳定,且upLOC的稳定性优于FBOX,而TOC75、SDH、DMWD在马尾松和杂种松球果发育不同时期中均表达不稳定,尤其是TOC75的表达稳定性最差。因此,upLOC和FBOX可作为内参基因,为今后的马尾松与杂种松功能基因调控验证研究提供实验基础。

关键词:马尾松;qRT-PCR;内参基因;球果

Evaluation of reference genes for qRT-PCR in Pinus massoniana and its introgressive hybrid cones of different development stages

ABSTRACT

Pinus massoniana Lamb. and P. hwangshanensis Hsia. are mainly distributed in southern China. The former is an important tree species of afforestation and economic forest, while the latter is an ornamental tree species in high altitude area. In the middle and lower reaches of the Yangtze river, P. massoniana mainly grows in the areas below 700m (above sea level), while P. hwangshanensis mostly distributes in the areas above 900m (above sea level). In the areas between 700 and 900m (above sea level), an introgressive hybrid of them exists, which is called hybrid pine in this paper. In this study, cones of P. massoniana and hybrid pine at different development stages were used as experimental materials.

According to former studies of reference genes, as well as applying sequence alignment on common sequences of reference genes and transcriptome data, eight genes were assigned as candidate reference genes. These genes are as follows: actin (ACT), protein TOC75-3 (TOC75), ubiquitin (UBQ), phosphoglycerate kinase 1 (PGK1) and succinate dehydrogenase (SDH), dystrophia myotonica WD repeat-containing Protein (DMWD), uncharacterized protein LOC103705956 (upLOC) and f-box family protein (FBOX). All these eight candidate reference genes, were characterized by qRT-PCR processing and the expression stability of candidate reference genes were evaluated by three software applications (geNorm, NormFinder and BestKeeper). The results showed that upLOC and FBOX were both expressed stable in different development stages of P. massoniana and hybrid pine cones, and the stability of upLOC was better than that of FBOX. The expression of TOC75, SDH and DMWD was unstable in different development stages of P. massoniana and hybrid pine cones, among them the TOC75 was the most unstable one. Therefore, upLOC and FBOX can be used as reference genes to provide experimental basis for the analysis and research on the expression regulation of functional genes in Pinus massoniana and hybrid pine in further study.

Key words: Pinus massoniana; qRT-PCR; reference genes; cones

目录

1 前言 1

1.1 内参基因的概述 2

1.1.1 内参基因的研究背景 2

1.1.2 内参基因的研究动态 3

1.2 内参基因的筛选方法 4

2 材料与方法 6

2.1 试验材料 6

2.2 主要仪器 6

2.3 RNA提取 6

2.4 RNA质量检测 7

2.5 cDNA合成 7

2.6 内参基因选择及引物设计 8

2.7 qRT-PCR反应 9

2.8 候选内参基因变异系数、扩增效率和稳定性分析 9

3 结果与分析 10

3.1 候选内参基因引物扩增效率和特异性分析 10

3.2 geNorm 12

3.3 NormFinder 13

3.4 BestKeeper 14

4 讨论 16

5 结论 18

6 致谢 19

参考文献 20

1 前言

qRT-PCR技术因其具有的高灵敏度、高特异性和重复性好等优点,已经被广泛用于生命科学的各个研究领域[1]。在进行qRT-PCR实验时,必须选用适宜的内参基因进行校正和标准化。目前,常用的内参基因有ACT(actin)、18SrRNA(18S ribosomal RNA)、GAPDH(glyceraldehyde-3-phosphate dehydrogenase)、EF-1α(transcription elongation factors)、TUA(α-tubulin)和TUB(β-tubulin)等。虽然从理论上讲,这些内参基因可以在任何组织及生理状态下稳定地表达,但实际研究表明,不同物种、不同生理状态以及不同试验条件下,内参基因的表达会发生变化,并不是始终稳定的。因此,在开展研究时,要根据实际情况选择适宜的内参基因。

马尾松(Pinus massoniana Lamb.)属于松科(Pinaceae)松属(Pinus),高度可达到45m左右,种子长卵圆形,4~5月开花,球果第二年10~12月成熟[2]。马尾松在我国的分布很广,南北都有分布,大多数分布于南方且遍布华中华南各地,一般生长在长江下游海拔600~700m以下、中游约1200m以上、上游约1500m以下的区域,是我国南部重要的用材树种之一,也是荒山造林的先锋树种。

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