Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction

Sun, Zhan‐Bin; Li, Shidong; Sun, Man-Hong

doi:10.1016/j.mimet.2015.05.009

Cited by 17 publications

(14 citation statements)

References 20 publications

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“…The level of transcription of Chi67 - 1 in the transformants was assayed using an IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, CA, USA) and SYBR Premix Ex Taq (Takara, Dalian, China), with the elongation factor EF1 as a reference gene (Sun et al 2015a). Primers Chi F and Chi R were designed and synthesized (Table 1), and the specificity was verified by using PCR amplification and sequencing.…”

Section: Methodsmentioning

confidence: 99%

Transformation of the endochitinase gene Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclerotiorum

et al. 2017

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Clonostachys rosea is a promising biocontrol fungus active against various plant fungal pathogens. In this study, the endochitinase-encoding gene Chi67-1, the expression of which is sharply upregulated in C. rosea 67-1 when induced by sclerotia, was transformed into the original isolate by protoplast transformation, and transformants were screened against Sclerotinia rot of soybean. The transformation efficiency was approximately 50 transformants per 1 × 107 protoplasts, and 68 stably heritable recombinants were assayed. The parasitic rates of 32.4% of the tested strains increased by more than 50% compared to 43.3% of the wild type strain in 16 h, and the Rc4-4 transformant showed a parasitic rate of 100% in 16 h. The control efficiencies of the selected efficient transformants to soybean Sclerotinia stem rot were evaluated in pots in the greenhouse, and the results revealed that Rc4-4 achieved the highest efficiency of 81.4%, which was 31.7% and 28.7% higher than the control achieved by the wide type and the pesticide carbendazim, respectively. Furthermore, the expression level of Chi67-1 was 107-fold higher in Rc4-4 than in the wild type, and accordingly, the chitinase activity of the recombinant increased by 140%. The results lay a foundation for the development of efficient genetically engineered strains of C. rosea.

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Section: Methodsmentioning

confidence: 99%

Transformation of the endochitinase gene Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclerotiorum

et al. 2017

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“…The gene tub was also used in the quantification of gene expressions in C. rosea when confronting with pathogenic fungi and plant nematodes (Mamarabadi et al., ; Zou, Tu, Liu, Tao, & Zhang, ). In our previous study, we found that EF1 was expressed most stably in C. rosea 67‐1 during parasitism on Sclerotinia sclerotiorum sclerotia, and we obtained a reliable expression profile of mycoparasitism‐related genes by using the reference gene EF1 (Sun, Li et al., ; Sun, Sun et al., ). In this study, analyses using geNorm, BestKeeper, and NormFinder showed that the expression of EF1 was not satisfactory during chlamydospore formation in C. rosea .…”

Section: Discussionmentioning

confidence: 94%

“…On the basis of the transcriptome data for C. rosea 67‐1 chlamydospore formation, a total of nine candidates were selected and cloned from the genome of C. rosea (Sun, Li et al., ; Sun, Sun et al., ), namely, ACT , EF1 , GAPDH , HIS , RPP , SSD , TBP , UBQ , and UCE (GenBank accession numbers: KP274072, KP274074‐KP274077, KX686112‐KX686115). The primers for the reference genes used for RT‐qPCR were designed using Primer Premier 6 (Table ), and their specificity was assessed with the following PCR program: 94°C for 3 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 20 s; final extension at 72°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…However, no reference gene is absolutely consistent in different organisms and their developmental stages; instead, the expression level of a reference gene is stable only under specific conditions. β ‐tubulin was used to normalize N‐acety1‐β‐D‐glucosaminidase gene in C. rosea when confronting with Fusarium culmorum (Mamarabadi, Jensen, & Lübeck, ), however, it was not available in mycoparasitic process (Sun, Li, & Sun, ; Sun, Sun, & Li, ). In Neurospora crassa , the expression of act was not affected by temperature, but it was inhibited under all‐dark conditions (Cusick et al., ).…”

Section: Introductionmentioning

confidence: 99%

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Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

Zhang

Sun

et al. 2017

MicrobiologyOpen

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Clonostachys rosea is a potential biocontrol fungus that can produce highly resistant chlamydospores under specific conditions. To investigate the genes related to chlamydospore formation, we identified reliable reference genes for quantification of gene expression in C. rosea 67‐1 during sporulation. In this study, nine reference genes, actin (ACT), elongation factor 1 (EF1), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), histone (HIS), RNA polymerase II CTD phosphatase Fcp1 (RPP), succinate‐semialdehyde dehydrogenase (SSD), TATA‐binding protein (TBP), ubiquitin (UBQ), and ubiquitin‐conjugating enzyme (UCE), were selected and cloned from 67‐1, and their expression stability during chlamydospore formation was determined using reverse transcription quantitative PCR and assessed using the software geNorm, NormFinder and BestKeeper. The Ct values of the candidates ranged from 19.9 to 29.7, among which HIS,ACT and SSD exhibited high expression levels. The statistical analysis showed that ACT and SSD were most stably expressed, while UBQ and GAPDH showed relatively large variations under different culture conditions. Calculation of pairwise variation value indicated that two reference genes were required for precise quantification. Finally, ACT and SSD were selected to normalize gene expression during chlamydospore production in C. rosea 67‐1. To the best of our knowledge, this is the first report of SSD as a reference gene. This study will facilitate the accurate quantification of differentially expressed genes during the generation of chlamydospores and contribute to the investigation of the molecular mechanism underlying chlamydospore formation in C. rosea.

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“…Interestingly, TUBA was shown to be a reliable reference gene for Penicillium expansum (De Clercq et al 2016) and Valsa mali var. mali ( Vmm ) (Yin et al 2013), and EF1 was found to be suitable for Clonostachys rosea (Sun et al 2015) and Tuber melanosporum (Cesare et al 2015). However, TUBA and EF1 were unstable and unsuitable for use as reference genes in Blumeria graminis (Pennington et al 2016), C. rosea (Sun et al 2015), and Pandora neoaphidis (Chen et al 2016).…”

Section: Discussionmentioning

confidence: 99%

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

Che

Shi

et al. 2016

AMB Expr

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Alternaria sp. MG1, an endophytic fungus isolated from Vitis vinifera, can independently produce resveratrol, indicating that this species contains the key genes for resveratrol biosynthesis. Identification of these key genes is essential to understand the resveratrol biosynthesis pathway in this strain, which is currently unknown in microorganisms. qRT-PCR is an efficient and widely used method to identify the key genes related to unknown pathways at the level of gene expression. Verification of stable reference genes in this strain is essential for qRT-PCR data normalization, although results have been reported for other Alternaria sp. strains. In this study, nine candidate reference genes including TUBA, EF1, EF2, UBC, UFD, RPS5, RPS24, ACTB and 18S were evaluated for expression stability in a diverse set of six samples representing different growth periods. We compared cell culture conditions and an optimized condition for resveratrol production. The comparison of the results was performed using four statistical softwares. A combination of TUBA and EF1 was found to be suitable for normalization of Alternaria sp. MG1 in different developmental stages, and 18S was found to be the least stable. The reference genes verified in this study will facilitate further research to explore gene expression and molecular mechanisms as well as the improvement of secondary metabolite yields in Alternaria sp. MG1. To our knowledge, this is the first validation of reference genes in Alternaria with the capability to produce resveratrol. Additionally, these results provide useful guidelines for the selection of reference genes in other Alternaria species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0283-z) contains supplementary material, which is available to authorized users.

show abstract

Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction

Cited by 17 publications

References 20 publications

Transformation of the endochitinase gene Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclerotiorum

Transformation of the endochitinase gene Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclerotiorum

Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

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