Effect Of Genetically Modified Chlamydomonas Feed On The Intestinal Flora Of Grouper

Objective:To study the preventive effect of transgenic feed on viral nervous necrosis of grouper and its effect on intestinal flora of grouper by using p CAMBIA1302-NNVCP and p Maa7IR-DGNNVIR transgenic Chlamydomonas reinhardtii.Methods:The study was divided into two parts,there are the preventive effect of transgenic Chlamydomonas reinhardtii feed on viral nervous necrosis of grouper and the effect of transgenic Chlamydomonas reinhardtii feed on intestinal flora of grouper.1.the preventive effect of transgenic Chlamydomonas reinhardtii feed on viral nervous necrosis of grouper:（1）transgenic Chlamydomonas reinhardtii and it’s identification:the p Maa7IR-DGNNVIR vectors were transferred into the chlamydomonas reinhardtii by glass bead method,the negative control was dd H₂O transferred to Chlamydomonas reinhardtii.The transformed algae strains were coated on a TAP medium containing 10μg/m L of paromomycin and extracted of DNA and RNA detecting by gel electrophoresis.（2）Enlargement of Chlamydomonas reinhardtii and production of feed:expanded culture of successfully transformed Chlamydomonas reinhardtii and collecting algae mud,mix with grouper feed in the ratio of algae mud:feed=1:1 to make genetically modified feed.（3）Collecting pearl gentian grouper with abnormal behavior of viral nervous necrosis,extract DNA and RNA to identify whether it carries neuronecrosis virus.Real-time PCR determines that the copy number of virus to injecting.（4）The preventive effect of transgenic Chlamydomonas reinhardtii mixing feed on neuronecrosis virus:200 pearl gentian grouper were divided into control group,CC425 feed group,p CAMBIA1302-NNVCP feed group and p Maa7IR-DGNNVIR feed group,50 tails per group,after 10 days of feeding,record the growth index of grouper;After injecting with21.67 copier/μL copy numbe of virus,Real-time PCR detection of NNV content in the brain tissue of grouper;48 h later,the pathological changes of eyeball and brain tissue of grouper were detected by pathological section;Record the death of grouper in each group,and the dead grouper was identified by PCR.2.Effects of transgenic Chlamydomonas reinhardtii affecting on intestinal flora of grouper:After feeding 10 days with relative feed,using 16 Sr RNA amplification sequencing to detect variation of intestinal flora in grouper on 24 h,48 h,72 h and 96 h,respectively.Results:1.the preventive effect of transgenic Chlamydomonas reinhardtii feed on viral nervous necrosis of grouper:（1）Transgenic Chlamydomonas reinhardtii and its identification:the algae strains transfected with p Maa7IR-DGNNVIR was at a concentration of 10μg/m L of paromomycin grew normally on the TAP medium,while the negative control algae strains died,Gel electrophoresis was used to detect the DNA and RNA of the transgenic Chlamydomonas reinhardtii,the results showed that p Maa7IR-DGNNVIR was successfully transferred into Chlamydomonas reinhardtii CC425.（2）Expansion culture and feed production of transgenic algae strains:after expanding the culture of Chlamydomonas reinhardtii with p CAMBIA1302-NNVCP and p Maa7IR-DGNNVIR expression vectors,the algae mud was collected with 145.4 g and170.6 g,respectively,according to the algae mud:feed the ratio of=1:1 is mixed to make genetically modified feed.（3）Detection of PCR identification results showed that the collected pearl gentian grouper carried nervous necrosis virus.Real-time PCR determines that the copy number of virus to injecting is 21.67 copier/μL.（4）The preventive effect of genetically modified Chlamydomonas reinhardtii mixed feed on nervous necrosis virus:after feeding for 10 days,the grouper in the control group gained an average weight of1.659 g,an average increase of 2.3 cm;the CC425 group gained an average of 1.989 g,an average increase of 2.8 cm;the average weight gain of the p Maa7IR/DGNNVIR group was2.018 g,an average increase of 2.9 cm;the average weight gain of the p CAMBIA1302-NNVCP group was 2.358 g,an average increase of 3.2 cm.Real-time PCR results showed that compared with the control group,the p CAMBIA1302-NNVCP group and the p Maa7IR-DGNNVIR group content of VNN in the brain tissue were significantly decreased compared with the control group.The expression of NNV in the p CAMBIA1302-NNVCP group and the p Maa7IR-DGNNVIR group were decreased by62.5%and 54.28%compare with control group,respectively,while the expression of NNV in the CC425 group had no significant difference compared with the control group.Pathological section results showed that the grouper eyeball tissue and brain tissue of the control group had obvious vacuolation.The CC425 group,p CAMBIA1302-NNVCP group and p Maa7IR-DGNNVIR group could inhibit the vacuolation of eyeball tissue and brain tissue.Statistics of grouper deaths in each group found that the mortality of the control group and CC425 group were significantly higher than of the p CAMBIA1302-NNVCP group and the p Maa7IR-DGNNVIR group,while the survival rate of the p CAMBIA1302-NNVCP group was decreased of 30%and 28%on control group and CC425group.The survival rate of the p Maa7IR-DGNNVIR group were decreased of 28%and 26%on the control group and the CC425 group.The survival rate of the CC425 group was increaesed 2%compare with the control group.The PCR method detected that the dead groupers the results showed that all groupers were carried the nervous necrosis virus.2.The effect of genetically modified Chlamydomonas reinhardtii feed on the intestinal flora of grouper:Analysis of sequencing data showed that after injecting with nervous necrosis virus,the number of OTUs in the control group and CC425 group were gradually decreased,while the number of OTUs in the p Maa7IR-DGNNVIR group and p CAMBIA1302-NNVCP group were gradually decreased increased;the Shannon indexes of CC425,p Maa7IR-DGNNVIR and p CAMBIA1302-NNVCP groups at different time points were significantly higher than of the control group,while the Simpson indexes of CC425,p Maa7IR-DGNNVIR and p CAMBIA1302-NNVCP groups at different time points were significantly decreased of the control group,indicating that the grouper gut microbial diversity in the CC425,p Maa7IR-DGNNVIR and p CAMBIA1302-NNVCP groups were higher than of the control group;At the phylum classification level,the dominant flora of each experimental group were Proteobacteria,Actinobacteria,Planctomycetes and Fusobacteria,and the relative abundance of Proteobacteria in each experimental group was increased significantly over time,and the relative abundance of Actinobacteria was showed a downward trend;At the genus classification level shows that the relative abundance of Mycobacterium was decreased gradually,while the relative abundance of Phenylobacterium is opposite,its proportion was gradually increased.The relative abundance of Vibrio in each group was increased significantly,and then stabilized.After injecting with nervous necrosis virus,the relative abundance of Burkholderia was increased gradually as time went on.At the genus classification level,the dominant intestinal flora in each group was divided into two types over time.Conclusion:Transformed the p CAMBIA1302-NNVCP and the p Maa7IR-DGNNVIR gene Chlamydomonas reinhardtii feed has a protective effect on grouper viral neuronecrosis.Its protective effect may be through reducing the expression of NNV in the grouper and reducing the vacuolization effect of NNV on the eyeballs and brain tissue of grouper,and reduces the damage of the virus to the body of the grouper,increases the diversity of the intestinal microbes of the grouper,and thereby reduces the mortality of the grouper.