| Tetrastigma hemsleyanum (Tetrastigma hemsleyanum Diels & Gilg ex Diels) is a precious medicinal plant in China, and the whole plant can be used as medicine. Especially the bulbous root is the most valuable part. However, the natural slow growth rate of Tetrastigma hemsleyanum has resulted that it hardly meets the demand of market. Expansin widely exists in the plant cell wall, it can not only make elongation growth of the plant cell, but also adapt to the surrounding environment by regulating the flexibility of cell wall. The plant endophytic fungi exist in nature extensively and vary in kind, and they have built a mutual beneficial relationship in the long process of evolution. The endophytic fungi can influence on the growth and development of plant, and improve the stress resistance such as drought resistance, salt resistance, disease resistance and so on. They can also make effects on metabolites produced by plant.In this study, the expansin gene (Th-exp) cDNA sequence is cloned from Tetrastigma hemsleyanum. Then, the endophytic fungi from root tuber of Tetrastigma hemsleyanum are isolated, identified and observed. Last, the express of Th-exp gene and flavonoid contents as medicinal component are detected from the aseptic plantlet inoculated with the fermentation broths of these endophytic fungi. The results are as follows:(1) According to the conservative region sequences of expansin gene in GenBank database, one pair of degenerate primers was designed for RT-PCR. The conservative region fragment was first amplified by PCR from cDNA template of Tetrastigma hemsleyanum. The full-length sequence of expansin gene cDNA (named as Th-exp) was extended by RACE (rapid-amplification of cDNA ends). The full length of Th-exp is 782 bp and contains a 630 bp open reading frame, which encodes a protein of 209 amino acids (GenBank accession number KP693606). The protein encoded by Th-exp habours a typical expansin structure, including 8 cysteine domain in N terminal region,4 conserved tryptophan domains in C terminal region, and histidine function domain in intermediate region. Blast alignment showed that Th-exp is similar to expansin genes of Vitis vinifera and Tarenaya hassleriana. Semi-quantitative RT-PCR and fluorescence quantitative PCR indicated that Th-exp can express in leaf, stem, ordinary fine root and calabash-shaped root, while the expression level in calabash-shaped root was higher than those in other organs.(2) By tissue culture method, thirty-one endophytic fungi varied in color, texture, growth rate etc were obtained from the old root tuber of Tetrastigma hemsleyanum. By amplifying and analyzing of the ITS rDNA sequences, the fungi belonged to ten generas, which were Fusarium, Rhizopycnis, Colletotrichum, Cylindrocarpon, Emericellopsi, Penicillium, Plectosphaerella, Aspergillus tubingensis, Alternaria, Sarocladium strictum respectively. Additionally, the strain of 8.16-10-2 was uncultured fungus and the Fusarium was the dominant fungi. Making the paraffin sections of root tuber from Tetrastigma hemsleyanum and dyed by safranine-fast green, the results showed that most endophytic fungi distributed in the cortex and phloem scatteredly, colonized in a single or multiple contiguous cells in the host roots.(3) The aseptic plantlets of Tetrastigma hemsleyanum were cultured on MS medium supplied with fermentation broths of the endophytic fungi. Fluorescence quantitative PCR showed that the expression of Th-exp gene in Th-9.7-1, Th-9.7-2, Th-11.23-1 was upregulated, Th-9.7-2 expressed three times more than the control group, while the expression of Th-exp gene in Th-8.16-8, Th-8.16-13-1 was reduced. Howerver, Th-8.16-13-1 plant was no changes obviously. The flavonoid contents were detected by the method of UV spectrophotometry at 500 nm. The results showed that the strains of 9.7-1,9.7-5,8.16-8, and 11.23-1 could induce the contents of flavonoid. Among them, Th-9.7-5 which was the highest contained 39.6194 mg/g-DW, while Th-9.7-2, Th-8.16-13-1 were similar to the control. |
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