[Selection of q RT-PCR reference genes for Amomum tsaoko seeds during dormancy release].

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.

[Cloning and expression analysis of GGPPS gene from Panax notoginseng].

According to the transcriptome dataset of Panax notoginseng, the key geranylgeranyl pyrophosphate synthase gene (GGPPS) in terpenoid backbone biosynthesis was selected to be cloned. Using specific primer pairs combining with RACE (rapid amplification of cDNA ends) technique, the full-length cDNA sequence with 1 203 bp, which containing a 1 035 bp open reading frame, was cloned and named as PnGGPPS. The corresponding full-length DNA sequence contained 2 370 bp, consisted of 1 intron and 2 exons. The deduced protein PnGGPPS contained 344 amino acids and shared more than 73% identity with GGPPS from Ricinus communis and Salvia miltiorrhiza. PnGGPPS also had specific Aspartic acid enrichment regions and other conserved domains, which belonged to the Isoprenoid-Biosyn-C1 superfamily. The quantitative real-time PCR showed that PnGGPPS expressed in different tissues of 1, 2, 3 years old root, stem, leaf and 3 years old flower, and the expression level in 3 years old leaf was significant higher than that in other organs, which suggested that it might not only be involved in the regulation of the growth and development, but also be associated with the biosynthesis of chlorophyll and carotenoids, the development of chloroplast, the shade habit and the quality formation of P. notoginseng.

[Antagonism of Trichoderma spp. to fungi caused root rot of Sophora tonkinensis].

OBJECTIVE: To study the antagonism of Trichoderma spp. to fungi S9(Fusarium solani)which caused root rot of Sophora tonkinensis and discuss the further develop prospects of microbial biological control in soil-borne diseases on Chinese herbal medicines. METHODS: Antagonism of H2 (Trichoderma harsianum), M6 (Trichoderma viride) and K1 (Trichoderma koningii) to Fusarium solani were researched by growth rate and confront culture. And their mechanisms were discussed. RESULTS: H2 and M6 had obvious competitive advantage, the growth rate of which were 1.43-2.72 times and 1.43-1.95 times as S9 respectively. The space competitive advantage of K1 was relatively weak; the growth rate was slower than S9. The antagonism of three species of Trichoderma spp. to S9 was in varying degrees. The antagonism to S9 of M6 and H2 was better,the inhibition rate were 100% and 82.35% respectively, even cultivated S9 for three days in advance. And their inhibition indexes were both reached class I. The inhibition index and inhibition rate of K1 was respectively 46.36% and class IV. The Trichoderma spp. could cause S9 mycelium to appear some phenomenon just like fracture, constriction reduced, digestion, etc. which were observed under the microscope. CONCLUSION: Trichoderma harsianum and Trichoderma viride showed the further develop prospects in the fight against soil-borne disease on Chinese herbal medicines.

[Molecular identification of geminivirus inducing vein yellowing in Abelmoschus manihot].

OBJECTIVE: The virus isolate H was identified by molecular biology,it was collected from Abelmoschus manihot plant showing leaf curl,yellow vein symptoms in Guangxi Botanical Garden of Medicinal Plant. METHODS: The virus isolate H was observed in electron micrograph, and conformed detected by PCR using universal primer pair for the genus Geminivirus. RESULTS: The results indicated that all sequences homologous to the specific fragment belonged to the genus Begomovirus of the family Geminiviridae. There was the highest similarity shared 95% homology at nucleotide between the specific fragment and DNA-A of Emilia yellow vein virus isolates. CONCLUSION: These findings suggested that there was geminiviridea in Abelmoschus manihot, and the disease probably caused by Emilia yellow vein virus.