香樟齿喙象发育期点温度和有效积温的研究毕业论文_林学毕业论文

香樟齿喙象发育期点温度和有效积温的研究毕业论文

2021-04-19更新

摘 要

香樟齿喙象Pagiophloeus tsushimanus 是2014年在上海新发现的一种专性危害香樟的钻蛀性害虫。目前,上海市14 个行政区的香樟林均有该虫分布,且有扩散的趋势,对周边地区的香樟人工林存在潜在风险。香樟齿喙象内参基因的筛选研究未有报道,因此,本文详细分析评估了不同发育阶段的的香樟齿喙象8个内参基因TBP、RPS27A、RPL、UBQ、GST、GAPDH、18s、RPS3的表达稳定性。研究结果如下:

香樟齿喙象转录组中选出的8个候选内参基因(18s、GAPDH、GST、RPL、RPS3、RPS27、TBP和UBP)的引物扩增溶解曲线均仅有显著的单一峰,表明选择的引物均可特异性地扩增各内参基因的对应产物,不存在引物二聚体。8个候选内参基因的引物均具有良好的扩增效率。RPS3的表达水平最高,TBP的表达水平最低,RPS27A在不同样品间的表达水平差异最大,GST表达水平在不同样品间差异最小。

geNorm软件分析结果显示,香樟齿喙象不同发育阶段的最稳定内参基因为TBP和 UBQ,同时RPL也是稳定的内参基因。本实验中Vn/n 1值均超过程序推荐值0.15,表明进行基因表达分析时需加入第3个内参基因进行数据校正。normFinder软件分析结果,用于不同发育阶段之间的最佳内参基因组合为18s and TBP,RPS3是推荐的最佳内参基因。Bestkeeper软件分析结果,RPS3、UBQ与TBP是较为稳定的内参基因。RefFinder在线综合分析结果,RPS3、UBQ与TBP3个内参基因在香樟齿喙象不同发育阶段具有较高的表达稳定性。

综上所述,在香樟齿喙象不同发育阶段进行基因表达分析时,可选择RPS3、UBQ与TBP作为内参基因进行数据校正。

关键词: 香樟齿喙象;内参基因;geNorm;normFinder;Bestkeeper;RefFinder

Study on the selection of reference genes in Pagiophloeus tsushimanus Morimoto

Abstract

Pagiophloeus tsushimanus, the beak of camphor camphor camphor, is a new bore-eating insect found in 2014. At present, in all the 14 administrative regions of Shanghai, there is a potential risk of the spread of this insect in the surrounding area. Camphor tooth beak like internal gene screening studies did not report, therefore, in this paper, detailed analysis to evaluate the different development stages of camphor tooth beak like eight inside the TBP genes, RPS27A, RPL, UBQ, GST, the expression of GAPDH and 18 s, RPS3 stability. The research results are as follows:

Camphor tooth beak like transcriptome selected eight internal candidate genes (18 s, GAPDH, GST, RPL, RPS3, RPS27, the TBP and UBP) primers amplification of dissolution curve were only significant single piece, show that selected primers are specific to amplify the corresponding internal gene product, there is no primer dimers. The primers of 8 candidate endogenous genes have good amplification efficiency. RPS3 has the highest expression level, TBP has the lowest expression level, RPS27A has the highest expression level difference among different samples, and GST has the lowest expression level difference among different samples.

According to geNorm software, TBP and UBQ were the most stable internal reference genes of camphor camphor rostris at different developmental stages, and RPL was also a stable internal reference gene. In this experiment, Vn/n 1 values exceeded the recommended value of the program by 0.15(FIG. 3), indicating that the third internal reference gene should be added for data correction in gene expression analysis. According to the analysis results of normFinder software, the optimal combination of 18s and TBP for different developmental stages is RPS3, which is recommended as the best endogenous gene. Bestkeeper software analysis showed that RPS3, UBQ and TBP were relatively stable internal reference genes. The results of RefFinder online comprehensive analysis showed that RPS3, UBQ and TBP3 internal reference genes had high expression stability at different developmental stages of camphor rostral elephant.

In conclusion, RPS3, UBQ and TBP can be selected as internal reference genes for gene expression analysis at different developmental stages of camphor rostral.

Key words: camphor rostral; Internal reference gene; GeNorm; NormFinder; Bestkeeper; RefFinder

目 录

1 绪论 1

1.1 引言 1

1.2 香樟齿喙象的研究现状 1

1.3 内参基因的研究 2

1.3.1 内参基因概述 2

1.3.2 内参基因的筛选方法 3

1.3.3 内参基因表达稳定性评估方法 3

1.3.4 内参基因的选择以及在昆虫领域应用 3

1.4 本研究的目的和意义 4

2 实验材料与方法 5

2.1 实验材料 5

2.1.1 供试昆虫 5

2.2 不同虫态总RNA提取和cDNA第一链合成 5

2.2.1 RNA的提取 5

2.2.2 cDNA的合成 6

2.3 候选内参基因的筛选及基因克隆测序 7

2.3.1 PCR反应 7

2.3.2 胶回收 8

2.3.3 基因克隆 8

2.4 候选内参基因稳定性的评价 9

2.4.1 RT-qPCR方法 9

2.4.2 数据处理与分析方法 9

3 结果与分析 10

3.1 香樟齿喙象8个内参基因分析 10

3.2 香樟齿喙象8个内参基因引物扩增效率和特异性 11

3.3 香樟齿喙象不同虫态8个候选内参基因的表达差异分析 12

3.4 香樟齿喙象不同虫态8个候选内参基因的表达稳定性分析 13

3.4.1 geNorm软件分析 13

3.4.2 normFinder软件分析 14

3.4.3 BestKeeper软件分析 15

3.4.4 RefFinder在线综合分析 16

4 结论与讨论 17

致 谢 19

参考文献 20

1 绪论

1.1 引言

香樟Cinnamomum camphora属于樟目Laurales樟科Lauraceae樟属Cinnamomum,常绿乔木,且形态美观,枝叶茂密,具有较高的经济价值和观赏价值,广泛分布于我国华南地区,是我国特产的珍贵木材和经济林树种[1]。由于大面积营造香樟人工纯林,林分结构简单,抵抗虫害能力弱,易受各种害虫危害,如樟巢螟Orthaga achatina、樟翠尺蛾Thalassodes quadraria、樟个木虱Trioza camphorae、橄绿瘤丛螟Orthaga olivacea[2],于2014年新发现一种专性危害香樟的钻蛀性害虫香樟齿喙象Pagiophloeus tsushimanus,属于鞘翅目Coleptera象虫科Curculionidae 魔喙象亚科Molytinae 树皮象族Hylobiini 齿喙象属Pagiophloeus,被确定为中国新记录种,国内仅分布于中国上海市与福建省福州市[3],国外分布于日本九州对马岛[4]。正模与副模均为日本对马岛的香樟上采集[4]。由于模式标本产地与我国华东地区地理区系相似,因此推测该虫是中国本地物种[3]。目前,上海市14 个行政区的香樟林均有该虫分布,且有扩散的趋势,对周边地区的香樟人工林存在潜在风险[5]

1.2 香樟齿喙象的研究现状

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