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新疆塔克拉玛干7株沙漠藻的分子鉴定及生物学特性研究

Identification and Biological Characteristics of Seven Algae in Taklimakan Desert of Xinjiang

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【Author in Chinese】 卡丽比努尔·艾依提

【Supervisor】 努尔古丽·热合曼

【Author's Information】 新疆师范大学, 微生物学, 2017, 硕士

【Abstract in Chinese】 本研究从新疆塔克拉玛干沙漠分离纯化的4株绿藻和3株蓝藻进行形态观察、分子生物学鉴定法来鉴定沙漠藻的遗传位置,探讨沙漠藻的生长周期并外界因素对藻类生长的影响分析藻种的最佳生长条件。通过高效液相色谱法和普通PCR基因检测法,分析沙漠藻藻毒素MC-LR和藻毒素合成酶基因mcyE基因并对新疆塔克拉玛干沙漠藻类分子生物学研究和藻类食品应用方面提供理论依据。首次,通过光学显微镜和扫描电镜观察从塔克拉玛干沙漠分离出来的7株藻类中3YS16(1)的是绿色、粘稠、油性,细胞较小,椭圆形藻;KXII(2)是黄色、干燥、难挑取,细胞偏大,球状藻;3YS21-1是绿色,油性,球状藻;YS2-3是偏黄色、干燥、难挑取、近球状藻。蓝藻BYS14-1、YS3-1和JP4-1是念状藻。其次,采用分子生物学方法18S rRNA和16S rRNA基因序列分析结果表明:沙漠绿藻3YS16(1)和3YS21-1的测序序列与GenBank上的Chlamydomonas incerta相似率是99%。YS2-3的测序序列与Chlorosarcinopsis communis相似率是97%。KXII(2)测序序列与Chlorosarcinopsis bastropiensis相似率是99%。沙漠蓝藻BYS14-1的测序序列与Aphanizomenon aphanizomenoides(丝囊藻)相似率是98%,YS3-1是与Leptolyngbya sp(鞘丝藻)相似率是99%,JP4-1的测序序列与GenBank已知序列同源性低,相似率95%。再次,绘制出7株沙漠藻的生长曲线并外界因素对藻类生长的影响结果表明,4株绿藻培养第3天开始进入对数生长期;3株蓝藻里面BYS14-1第2天进入对数生长期;YS3-1第3天进入对数生长期;JP4-1在第11天进入对数生长期。7株沙漠藻在培养的一月没没有进入衰亡期。3YS16(1)的最适生长的pH值为6.0,温度为40℃,光强度为2500 Lux;3YS21-1的最适生长的pH值为5.0,温度为40℃,光强度为2500Lux;KXII(2)的最适生长的pH值为8.0,温度为35℃,光强度为4500 Lux;YS2-3的最适生长的pH值为8.0,温度为30℃,光强度为2500 Lux;BYS14-1的最适生长的pH值为5.0,温度为30℃,光强度为3500 Lux;YS3-1的最适生长的pH值为5.0,温度为30℃,光强度为2500 Lux;JP4-1的最适生长的pH值为6.0,温度为40℃,光强度为4500 Lux。最后,通过高效液相色谱法(HPLC)和普通PCR基因检测法检测微囊藻毒素MC-LR和藻毒素合成酶mcyE基因,HPLC实验结果表明微囊藻毒素标准品保留时间为17.8 min,但7株沙漠藻此处没有保留。微囊藻毒素mcyE基因检测结果表明mcyE基因引物没有扩增出来7株沙漠藻E基因序列,可知这7株藻没有控制产生藻毒素的合成酶基因,说明7株藻不产生藻毒素。通过普通PCR检测mcyE基因的实验结果跟高效液相色谱法检测微囊藻毒素MC-LR的结果一致。说明从塔克拉玛干沙漠分离纯化的这些藻不产生藻毒素。

【Abstract】 The study isolated 4 strains green algae and 3 strains cyanobacteria from Xinjiang Taklimakan desert to identify genetic position by morphological and molecular biological method,to investigate the growth cycle and effects of temperature,light intensity and pH value on the growth of desert algae.analysis MC-LR and microcystin synthetase mcyE gene of desert algae by method of high performance liquid chromatography and detection gene,to provide a theoretical basis for molecular biology and application of algae foods of Xinjiang Taklimakan desert algae.First,The 3YS16(1)is divided into oval algae by optical microscope and electron microscop,3YS21-1、YS2-3 、KXII(2)into spherical algae;3YS16 is green,thick,oily,small cells and oval algae;KXII(2)is yellow,dry,hard to pick,cells are relatively large,spherical algae;3YS21-1 is green,oily,spherical algae;YS2-3 is yellow,dry,hard to pick,nearly spherical algae.BYS14-1、YS3-1 and JP4-1is Nostoc.Secondly,Molecular biology methods18 S rRNA and 16 S rRNA gene sequence analysis demonstrated that strains 3YS16(1)and 3YS21-1 were identified Chlamydomonas incerta.strain YS2-3 were identified Chlorosarcinopsis communis.strain KXII(2)were identified Chlorosarcinopsis bastropiensi.Strain BYS14-1 were identified Anabaena.Strain YS3-1 were identified Leptolyngbya sp.low homology of sequencing JP4-1 with known GenBank sequence and similarity rate is 95%,so may be JP4-1 is new genus.Again,draw out seven strains of desert algae growth curve and study the effects of external factors on the desert algae.The results showed that 4 strains of algae of the third day entered the logarithmic growth phase.cyanobacteria of BYS14-1 second days into the logarithmic growth phase;YS3-1 third days into the logarithmic growth phase;JP4-1on the ninth day after entered the logarithmic period,seven desert algae did not enter the decline phase.Desert algae 3YS16(1)suitable pH vales is the acidic environment,and the optimum pH was 6.0,temperature is 40℃,light intensity is 2500 Lux;3YS21-1 suitable pH was 5.0,temperature is 40℃,light intensity is 2500 Lux;KXII(2)optimum pH was 8.0,temperature is 35℃,light intensity is 4500 Lux;YS2-3 optimum pH was 8.0,temperature is 30℃,light intensity is 2500 Lux;BYS14-1 optimum pH was 5.0,temperature is 30℃,light intensity is 3500 Lux;YS3-1 optimum pH was 5.0,temperature is 30℃,light intensity is 3500 Lux;JP4-1 optimum pH was6.0,temperature is 40℃,light intensity is4500 Lux.Finally,by high performance liquid chromatography(HPLC)and PCR gene detection method for microcystin MC-LR and microcystin synthetase gene mcyE,HPLC results showed that microcystin standard retention time is 17.8min,but 7 strains desert algae without reservation.The PCR gene detection results showed that primers did not amplified the E gene sequences.The results showed that these 7 strains of desert algae have not microcystin synthetase gene,indicating that the 7 strains of algae did not produce algal toxins.The results were consistent with the results of high performance liquid chromatograpHy for detection of microcystin MC-LR.The results showed that the algae isolated from the Taklimakan Desert did not produce algal toxins.

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