Peer Review #3 of "Evaluation of potential reference genes for quantitative RT-PCR analysis in spotted sea bass (Lateolabrax maculatus) under normal and salinity stress conditions (v0.1)"

The aim of this study was to select the most suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) of spotted sea bass (Lateolabrax maculatus), an important commercial marine fish in Pacific Asia, under normal physiological and salinity stress conditions. A total of 9 candidate reference genes (HPRT, GAPDH, EF1A, TUBA, RPL7, RNAPol II, B2M, ACTB and 18S rRNA) were analyzed by qRT-PCR in 10 tissues (intestine, muscle, stomach, brain, heart, liver, gill, kidney, pectoral fins and spleen) of L. maculatus. Four algorithms, geNorm, NormFinder, BestKeeper, and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. The results showed the 18S rRNA was most stable in different tissues under normal conditions. During salinity stress, RPL7 was the most stable gene according to overall ranking and the best combination of reference genes was RPL7 and RNAPol II. In contrast, GAPDH was the least stable gene which was not suitable as reference genes. The study showed that different algorithms might generate inconsistent results. Therefore, the combination of several reference genes should be selected to accurately calibrate system errors. The present study was the first to select reference genes of L. maculatus by qRT-PCR and provides a useful basis for selecting the appropriate reference gene in L. maculatus. The present study also has important implications for gene expression and functional genomics research in this species or other teleost species.

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Cloning and function analysis of BAG family genes in wheat

Functional Plant Biology ◽

10.1071/fp15317 ◽

2016 ◽

Vol 43 (5) ◽

pp. 393

Author(s):

Shiming Ge ◽

Zhen Kang ◽

Ying Li ◽

Fuzhen Zhang ◽

Yinzhu Shen ◽

...

Keyword(s):

Heat Stress ◽

Salinity Stress ◽

Heat Tolerance ◽

Expressed Sequence Tag ◽

Function Analysis ◽

Pcr Analysis ◽

Subcellular Localisation ◽

Hybrid Technique ◽

Rt Pcr ◽

Conserved Domain

By analysing the cDNA microarray of the salt tolerant mutant of wheat RH8706–49 under salinity stress, our results showed an expressed sequence tag fragment and acquired an unknown gene (designated as TaBAG) with a BAG conserved domain through electronic cloning and RT–PCR technology. The gene was registered into GenBank (No. FJ599765). After homologous alignment analysis, electronic cloning, and amplifying with RT–PCR, the other gene with a BAG conserved domain, TaBAG2, was obtained and registered into GenBank (No. GU471210). Quantitative PCR analysis demonstrated that TaBAG2 expression was induced by saline and heat stress. TaBAG gene expression under salinity stress increased remarkably but showed an insignificant response to heat stress. The adversity stress detection results showed that Arabidopsis overexpressing TaBAG and TaBAG2 exhibited an obvious salt tolerance increase. Under heat stress, Arabidopsis overexpressing TaBAG2 showed increased heat tolerance; however, the heat tolerance of Arabidopsis overexpressing TaBAG did not vary significantly under heat stress. Subcellular localisation results demonstrated that TaBAGs were mainly located in the cytoplasm and the cell nucleus. We applied fluorescence complementation and yeast two-hybrid technique to prove that TaBAG2 can obviously bond with TaHsp70 and TaCaMs. After the respective mutation of aspartic acid (D) and arginine (R) at high conservation in BAG domain of TaBAG2, the bonding interaction between TaBAG2 and TaHsp70 disappeared, indicating that the two amino acids were the key loci for the interaction between TaBAG2 and TaHsp70. Heat tolerance detection results demonstrated that the heat tolerance of Arabidopsis overexpressing and cotransfected with TaBAG2 and TaHsp70 was much higher than that of Arabidopsis overexpressing TaBAG2 and Arabidopsis overexpressing TaHSP70. This finding implies that the synergistic use of TaBAG2 and TaHSP70 can improve heat tolerance of plants.

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