3.3 Identification and expression of the genes in ...
Because the genes LDC，CAO，PKS，BBE，P450 and 2OGD had been proposed to participate in the Hup A biosynthetic pathway in plants［5］，we examined these genes and their expression in the two endophytic fungi.These included 1 LDC gene，6 CAO genes，27 PKS genes，2 BBE genes，111 P450s，and 35 2OGDs in hy4，while Cg01 included 2 LDC genes，12 CAO genes，47 PKS genes，4 BBE genes，280 P450s，and 39 2OGDs.Both hy4 and Cg01 are capable of producing Hup A，indicating hy4 and Cg01 may have shared Hup A-biosynthesis genes，which are orthologous in these two fungi or clustered together in the phylogenetic analysis.Therefore，we have done orthologous analysis as well as phylogenetic analysis between hy4 and Cg01.
Within the LDC genes，Pp LDC，Cg LDC1，and Cg LDC2 were orthologues.The sole LDC gene(Pp LDC)in hy4 was down-regulated.The homologous Cg LDC1 was up-regulated while Cg LDC2 was down-regulated.
There were 7 CAO genes in Cg01 which had orthologous genes in hy4.In addition，in the phylogenetic tree of CAO，CgCAO5 clustered with PpCAO1 and Shiraia sp.slf14，which has been reported to produce Hup A(Zhu et al.，2010).In these 8 CAO genes，4 were downregulated in the induced group and 3 showed low expression(NOTEST).The expression of the remaining one(CgCAO4)was also low with no significance difference between the two groups(FPKM=0.6～0.9).For the orthologues of CgCAO4 in hy4，one was downregulated and one did not show a significantly different expression between the two groups.Considering that CAO was proposed as the prophase skeleton synthase in Hup A，the induced Hup A increase might be more closely related to later modification.Therefore，CgCAO4 was considered the candidate biosynthesis gene.
There were 27 and 47 PKSgenes(including PKS-NRPShybrids)in hy4 and Cg01，respectively.Almost all PKSs in the two endophytic fungi were type I，except Cg-PKS40 which was typeⅢ.Though there were more PKS genes in Cg01 than in hy4，most of the PKS genes in Cg01(including hybrid，37/47)were seldom expressed(NOTEST)，and three were down-regulated，one up-regulated，and the others were not differentially expressed.PKSs with indispensable domains and orthologues between hy4 and Cg01 were the center of our focus.From the known chemical structure of Hup A，PKS that biosynthesize Hup A should only contain AT(acyl transferase)，KS(ketosynthase)，ACP(acyl carrier protein)，and/or TE(thioesterase)domains.Only 3 PKS had these indispensable domains in hy4 and Cg01.These genes were either NOTEST or down-regulated in the DEG analysis in both fungi.A total of 15 gene pairs were orthologues，including one pair clustering together in the phylogenetic tree between hy4 and Cg01，all of which were NOTEST or down-regulated in either fungus.Therefore，no candidate PKS genes were obtained for further validation by these two methods.We thus selected the upregulated Cg PKS35 and relatively highly expressed PKS genes Cg PKS14 and Cg PKS21 as the candidates for validation.(https://www.daowen.com)
All BBE genes in hy4 showed low expression(NOTEST)，while in Cg01 3 were downregulated and 1 showed low expression(NOTEST).Thus，we proposed that these identified BBE do not likely participate in Hup A biosynthsis.
We identified 111 P450 genes in hy4 and 280 in Cg01.In both endophytic fungi，the superfamily with the most genes was CYP65，with 109 and 203 genes in hy4 and Cg01，respectively.A total of 35 and 39 2OGD sequences were detected by searching for the 2OG-FeII_Oxy motif against the whole genomes of hy4 and Cg01，respectively.As most biosynthetic genes for SMs were clustered together，we identified the genomic locations of putative P450 and 2OGD genes to explore gene clustering.We regarded 2 P450s，2 2OGDs，one P450 and one 2OGD located within 10 genes as a gene cluster.There were 15 and 43 P450 gene clusters in hy4 and Cg01，respectively，with hy4_CYP_09247-RA and hy4_CYP_09249-RA up-regulated，and no P450 cluster upregulated in Cg01.The 2OGD gene clusters were fewer，with 3 and 4 in hy4 and Cg01，respectively.However，no 2OGD gene clusters were upregulated after induction.In the gene clusters of containing both P450 and 2OGD genes，there was no cluster with all genes upregulated.Most showed low expression(NOTEST)，or one upregulated while others showed no detectable difference in expression.
Taken together，we listed the LDC genes Cg LDC1 and Cg LDC2，the CAO gene CgCAO4，and the PKS genes Cg PKS35，Cg PKS14，and Cg PKS21 as candidate Hup A biosynthesis genes for validation.