毛脉酸模内生真菌体外抗肿瘤活性菌株筛选及其生物学特性研究
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摘要
毛脉酸模(Rumex gmelini Turcz.)为蓼科酸模属植物。民间以根入药,对淋病、上呼吸道感染性疾病、癣病和疮毒有确切疗效,具有抗肿瘤、抗真菌、镇咳平喘、降压、抗病毒和抗氧化等作用。内生真菌(endophytic fungus)是指生活在健康植物的各种组织和器官内部的真菌,被感染的植物不表现出明显的外在病症,可通过组织学方法、从严格表面消毒的植物组织中分离的方法以及从植物组织内直接扩增出微生物DNA的方法来证明其内生性。本文对毛脉酸模内生真菌进行了以下研究：
(1)毛脉酸模内生真菌体外抗肿瘤活性菌株的筛选
本实验用分离得到的16株毛脉酸模内生真菌对LOVO、SGC-7901、MCF-7、SMMC-7721、MGC-803、A549、SK-OV-3、HeLa 8株人体肿瘤细胞进行了体外抗肿瘤实验。运用MTT法进行实验,记录结果,采用SPSS16.0软件进行分析。筛选得到了4株抗肿瘤效果的内生真菌,其中菌RGT-S12对MCF-7,LOVO, SK-OV-3细胞具有显著的抑制作用。菌RGT-S31对A549,MGC-803,HeLa细胞具有显著的抑制作用。菌RGT-S42对SGC-7901,LOVO,SK-OV-3细胞具有显著的抑制作用。菌RGT-S44对SMMC-7721,MGC-803,A549和HeLa细胞具有显著的抑制作用。
(2)对筛选得到的毛脉酸模内生真菌进行分子鉴定
通过对所筛选出的3株菌进行rDNA扩增、测序及遗传进化的分析,并结合前期基础的形态鉴定,结果表明,菌RGT-S31为镰刀孢属Fusarium oxysporum,菌RGT-S42小尾孢属(Cercosporella)真菌,菌RGT-S44为镰刀属uncultured Fusarium真菌。
(3)筛选得到的毛脉酸模内生真菌生物学特性研究
通过改变不同培养条件对所筛选出的4株内生真菌进行培养特性研究,结果表明：菌RGT-S12在PDA培养基生长最快；最适宜PH值为6,最适温度为30℃；可以很好的利用乳糖和淀粉为碳源,利用KNO3为氮源。菌RGT-S31在四种培养基上均能较好的生长；最适生长pH值为pH8,最适温度为30℃；可以很好的利用麦芽糖为碳源,利用KNO3为氮源。菌RGT-S42在基本培养基能较好的生长,在查氏培养基上生长缓慢；的最适生长pH值为pH5,最适温度为25℃；可以很好的利用木糖为碳源,利用蛋白胨和NH4NO3为氮源。菌RGT-S48在PDA培养基能较好的生长,在查氏培养基上生长缓慢；最适生长pH值为pH 7,最适温度为30℃；可以很好的利用果糖为碳源,利用NaNO3为氮源。
本实验进行了毛脉酸模内生真菌体外抗肿瘤实验,筛选出4株有体外抗肿瘤效果的内生真菌,并对筛选得出的内生真菌进行了鉴定和培养特性研究,为开发毛脉酸模内生真菌资源和寻找新的抗肿瘤药物提供依据。
Rumex gmelini Turcz. belongs to the family of Polygonaceae. Its root and rhizome have been used for antifungal, antitumor, antitussive and expectrant, antiasthma, antivirus and antioxidation. Endophytic living in a healthy plant is a variety of tissues and organs within the fungus infected plants do not show obvious external symptoms. We may proved their endogenous by separation from strict surface disinfectant plant tissue and direct amplification of DNA from plant tissues. The results are as follows.
(1) Rumex gmelini endophyte strains in vitro screening of anti-tumor activity
In this experiment, sixteen strains of endophytic isolated from R. gmelini were tested on LOVO, SGC-7901, MCF-7, SMMC-7721, MGC-803, A549, SK-OV-3, HaLe eight strains of human tumor cells in vitro for antitumor. MTT was used in this experiment, and SPSS16.0 was used to analyze data. Four strains of endophytic were proved anti-tumor effect, RGT-S12 on MCF-7, LOVO, SK-OV-3 cells significantly inhibited; RGT-S31 on A549, MGC-803, HeLa cells significantly inhibited; RGT-S42 on SGC-7901, LOVO, SK-OV-3 cells significantly inhibited; RGT-S44 on SMMC-7721, MGC-803, A549, HeLa cells significantly inhibited.
(2) Molecular identification of endophytic from R. gmelini
We selected three strains which have antitumor effect, and identified them by sequencing their ITS, the results show that:the RGT-S31 strain is Fusarium Oxysporum; the RGT-S42 strain is Cercosporella; the RGT-S44 strain is Uncultured Fusarium.
(3) Biological characteristics of Endophyte from R. gmelini
The cultural characteristics of four selected strains of endophytic were studied by changing the different culture conditions. The results show that: RGT-S12 grow fastest in PDA medium; the most suitable PH value 6, the optimum temperature is 30℃; can be a good use of lactose and starch as carbon source, using KNO3 as nitrogen source. RGT-S31 could better growth medium in the four kinds of bacteria; optimum growth pH value 8, the optimum temperature is 30℃; can be a good use of maltose as carbon source, using KN03 as nitrogen source. RGT-S42 have a good growth in the basic medium while having a slow-growing in the investigation's; the optimum growth pH value 5, the optimal temperature was 25℃; can be a good use of xylose as the carbon source, use of peptone and NH4NO3 as nitrogen source. RGT-S44 can be good growth in the PDA medium while growth slowly in the investigation's; optimum growth pH value7, the optimum temperature is 30℃; can put to good use fructose as the carbon source, using NaNO3 as the nitrogen source.
The experiments have studies the anti-tumor effect of endophytic in vitro and found out four strains finally, then we made the identification and studied the cultural characteristics of them. The results of these studies can offer the theoretic and the practical guide to develop the resources of the endophytic fungi isolated from Rumex gmelini Turcz.
(1)毛脉酸模内生真菌体外抗肿瘤活性菌株的筛选
本实验用分离得到的16株毛脉酸模内生真菌对LOVO、SGC-7901、MCF-7、SMMC-7721、MGC-803、A549、SK-OV-3、HeLa 8株人体肿瘤细胞进行了体外抗肿瘤实验。运用MTT法进行实验,记录结果,采用SPSS16.0软件进行分析。筛选得到了4株抗肿瘤效果的内生真菌,其中菌RGT-S12对MCF-7,LOVO, SK-OV-3细胞具有显著的抑制作用。菌RGT-S31对A549,MGC-803,HeLa细胞具有显著的抑制作用。菌RGT-S42对SGC-7901,LOVO,SK-OV-3细胞具有显著的抑制作用。菌RGT-S44对SMMC-7721,MGC-803,A549和HeLa细胞具有显著的抑制作用。
(2)对筛选得到的毛脉酸模内生真菌进行分子鉴定
通过对所筛选出的3株菌进行rDNA扩增、测序及遗传进化的分析,并结合前期基础的形态鉴定,结果表明,菌RGT-S31为镰刀孢属Fusarium oxysporum,菌RGT-S42小尾孢属(Cercosporella)真菌,菌RGT-S44为镰刀属uncultured Fusarium真菌。
(3)筛选得到的毛脉酸模内生真菌生物学特性研究
通过改变不同培养条件对所筛选出的4株内生真菌进行培养特性研究,结果表明：菌RGT-S12在PDA培养基生长最快；最适宜PH值为6,最适温度为30℃；可以很好的利用乳糖和淀粉为碳源,利用KNO3为氮源。菌RGT-S31在四种培养基上均能较好的生长；最适生长pH值为pH8,最适温度为30℃；可以很好的利用麦芽糖为碳源,利用KNO3为氮源。菌RGT-S42在基本培养基能较好的生长,在查氏培养基上生长缓慢；的最适生长pH值为pH5,最适温度为25℃；可以很好的利用木糖为碳源,利用蛋白胨和NH4NO3为氮源。菌RGT-S48在PDA培养基能较好的生长,在查氏培养基上生长缓慢；最适生长pH值为pH 7,最适温度为30℃；可以很好的利用果糖为碳源,利用NaNO3为氮源。
本实验进行了毛脉酸模内生真菌体外抗肿瘤实验,筛选出4株有体外抗肿瘤效果的内生真菌,并对筛选得出的内生真菌进行了鉴定和培养特性研究,为开发毛脉酸模内生真菌资源和寻找新的抗肿瘤药物提供依据。
Rumex gmelini Turcz. belongs to the family of Polygonaceae. Its root and rhizome have been used for antifungal, antitumor, antitussive and expectrant, antiasthma, antivirus and antioxidation. Endophytic living in a healthy plant is a variety of tissues and organs within the fungus infected plants do not show obvious external symptoms. We may proved their endogenous by separation from strict surface disinfectant plant tissue and direct amplification of DNA from plant tissues. The results are as follows.
(1) Rumex gmelini endophyte strains in vitro screening of anti-tumor activity
In this experiment, sixteen strains of endophytic isolated from R. gmelini were tested on LOVO, SGC-7901, MCF-7, SMMC-7721, MGC-803, A549, SK-OV-3, HaLe eight strains of human tumor cells in vitro for antitumor. MTT was used in this experiment, and SPSS16.0 was used to analyze data. Four strains of endophytic were proved anti-tumor effect, RGT-S12 on MCF-7, LOVO, SK-OV-3 cells significantly inhibited; RGT-S31 on A549, MGC-803, HeLa cells significantly inhibited; RGT-S42 on SGC-7901, LOVO, SK-OV-3 cells significantly inhibited; RGT-S44 on SMMC-7721, MGC-803, A549, HeLa cells significantly inhibited.
(2) Molecular identification of endophytic from R. gmelini
We selected three strains which have antitumor effect, and identified them by sequencing their ITS, the results show that:the RGT-S31 strain is Fusarium Oxysporum; the RGT-S42 strain is Cercosporella; the RGT-S44 strain is Uncultured Fusarium.
(3) Biological characteristics of Endophyte from R. gmelini
The cultural characteristics of four selected strains of endophytic were studied by changing the different culture conditions. The results show that: RGT-S12 grow fastest in PDA medium; the most suitable PH value 6, the optimum temperature is 30℃; can be a good use of lactose and starch as carbon source, using KNO3 as nitrogen source. RGT-S31 could better growth medium in the four kinds of bacteria; optimum growth pH value 8, the optimum temperature is 30℃; can be a good use of maltose as carbon source, using KN03 as nitrogen source. RGT-S42 have a good growth in the basic medium while having a slow-growing in the investigation's; the optimum growth pH value 5, the optimal temperature was 25℃; can be a good use of xylose as the carbon source, use of peptone and NH4NO3 as nitrogen source. RGT-S44 can be good growth in the PDA medium while growth slowly in the investigation's; optimum growth pH value7, the optimum temperature is 30℃; can put to good use fructose as the carbon source, using NaNO3 as the nitrogen source.
The experiments have studies the anti-tumor effect of endophytic in vitro and found out four strains finally, then we made the identification and studied the cultural characteristics of them. The results of these studies can offer the theoretic and the practical guide to develop the resources of the endophytic fungi isolated from Rumex gmelini Turcz.
引文
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