Genome-wide identification of new reference genes for RT-qPCR normalization in CGMMV-infected Lagenaria siceraria.
Genome-wide identification of new reference genes for RT-qPCR normalization in CGMMV-infected Lagenaria siceraria.
复制标题
在 CGMMV 感染的 Lagenaria siceraria 中进行 RT-qPCR 标准化的新参考基因的全基因组鉴定。
作者：
Zhang C;Zheng H;Wu X;Xu H;Han K;Peng J;Lu Y;Lin L;Xu P;Wu X;Li G;Chen J;Yan F
狭叶裙带菜是一种重要的葫芦科作物，但在感染黄瓜绿斑驳花叶病毒(CGMMV)时，还没有确定合适的参考基因(RGS)。用RT-qPCR方法从葫芦叶片或果实的RNA-Seq数据集中筛选出16个叶和果实的候选RGS和18个候选RGS。使用geNorm、NormFinder、BestKeeper和RefFinder对这些基因的表达稳定性进行了测定和排序。综合分析，LsCYP、LsH3和LsTBP为葫芦叶的最佳RGS，LsP4H、LsADP和LsTBP为果实的最佳RGS。LsWD、LsGAPDH和LsH3对CGMMV侵染下的叶片和果实均有较好的药效。利用异戊烯基转移酶(IPT)和DNA引导的RNA聚合酶(DdRP)验证了从葫芦中鉴定出的最稳定的RGS对CGMMV的响应。所有候选RGS在RT-qPCR中的表现与转录组数据库中的数据一致。结果表明，LsWD、LsGAPDH和LsH3是叶片的最适内源RGS，LsH3、LsGAPDH、LsP4H和LsCYP是果实的最适内源RGS。
Lagenaria siceraria is an economically important cucurbitaceous crop, but suitable reference genes (RGs) to use when the plants are infected by cucumber green mottle mosaic virus (CGMMV) have not been determined. Sixteen candidate RGs of both leaf and fruit and 18 candidate RGs mostly from separate RNA-Seq datasets of bottle gourd leaf or fruit were screened and assessed by RT-qPCR. The expression stability of these genes was determined and ranked using geNorm, NormFinder, BestKeeper and RefFinder. Comprehensive analysis resulted in the selection of LsCYP, LsH3, and LsTBP as the optimal RGs for bottle gourd leaves, and LsP4H, LsADP, and LsTBP for fruits. LsWD, LsGAPDH, and LsH3 were optimal for use in both leaves and fruits under the infection of CGMMV. Isopentenyl transferase (IPT) and DNA-directed RNA polymerase (DdRP) were used to validate the applicability of the most stable identified RGs from bottle gourd in response to CGMMV. All the candidate RGs performed in RT-qPCR consistently with the data from the transcriptome database. The results demonstrated that LsWD, LsGAPDH and LsH3 were the most suitable internal RGs for the leaf, and LsH3, LsGAPDH, LsP4H and LsCYP for the fruit.
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DOI：
10.1042/bcj20161053
发表时间：
2017-05-04
期刊：
The Biochemical journal
影响因子：
--
作者：
Bortoluzzi A;Amato A;Lucas X;Blank M;Ciulli A
通讯作者：
Ciulli A
影响因子：
1.5
作者：
Ali, A;Natsuaki, T;Okuda, S
通讯作者：
Okuda, S
影响因子：
3.7
作者：
Kong Q;Yuan J;Niu P;Xie J;Jiang W;Huang Y;Bie Z
通讯作者：
Bie Z
DOI：
10.7235/hort.2015.14149
发表时间：
2015-04-01
影响因子：
--
作者：
Lee, Hee Ju;Kim, Mi-Kyeong;Chun, Changhoo
通讯作者：
Chun, Changhoo
影响因子：
9.3
作者：
Ceelen, Liesbeth;De Craene, Jurgen;De Spiegelaere, Ward
通讯作者：
De Spiegelaere, Ward