| Anthurium andraeanum is a perennial and evergreen herb of the family Araceae,which is popular because of its rich and gorgeous colors,peculiar appearance,rich lusters and long flowering period.Authura is a kind of important ornamental plant in the market which can be made into pot flowers,fresh cut flowers and landscape decorations.Authura is an ornamental plant with beautiful spathes and leaves,and its leaf color is also an important economic character that determines its ornamental value.Leaf color mutants are ideal materials for the study of plant pigment metabolism.Therefore,this study of authura's leaf color mutant has special theoretical significance and application value.In this study,using the wild-type authura 'Alabama' and its leaf color mutant produced by somatic cell mutations as the experiment materials,the mechanism of leaf color variation in authura mutant were analyzed by quantitative analysis of chlorophyll biosynthetic intermediate metabolites,transcriptome sequencing and quantitative real-time PCR.The main experimental results were as follows:1.The relative contents of chlorophyll and biosynthesis intermediates in 6 leaves were measured by utilizing wild-type authura with young leaves(CK-Y),mature leaves(CK-M),old leaves(CK-A)and leaf color mutant with young leaves(Mut-Y),mature leaves(Mut-M)and old leaves(Mut-A)as experimental materials.The results showed that,compared with the wild type,the chlorophyll content of leaf color mutant within young leaves,mature leaves and old leaves were reduced by 79.41%,75.37%and 48.59%respectively,and their ratios between chlorophyll a and chlorophyll b were 3.00:1.49,3.00:0.91 and 3.00:1.44,with an average of 3.00:1.28,while the average ratio of wild type was 3.00:1.28.In the young and mature leaves of the leaf color mutant,the content of protoporphyrin IX was reduced by 85.27%and 63.22%.In the old leaves,the coproporphyrinogen ? was reduced by 50.00%and the accumulation of its downstream intermediate metabolites also decreased correspondingly,all reaching significant levels(p<0.05),which eventually led to the reduction of chlorophyll synthesis and yellow leaves.In general,the chlorophyll content increased and the degree of leaf yellowing weakened gradually with the development of the leaves,and the leaves partially turned green during the old stage.2.A total of 175,951 transcripts and 78,763 unigenes were obtained by transcriptome sequencing and sequence assembly of wild type and leaf color mutant of authura,which are with an average length of943.53 bp and 699.46 bp respectively.In addition,12,415 unigenes were annotated in KOG database,and there were 3,260 unigenes annotated in KEGG database.According to the GO analysis,a total of 25,680 unigene gene sequences were annotated into 70 functional groups,including 21 molecular functions,27 biological processes,and 22 cellular components.3.According to the results of transcriptome sequencing,the significant difference genes were filtered by setting two times of difference,i.e.,|FoldChange|>2 and q-value<0.05 as the conditions.The observations are achieved as follows.Firstly,compared with the wild type,3,679 differentially expressed genes were screened between CK-Y and Mut-Y,with 2,550 genes up-regulated and 1,129 genes down-regulated in Mut-Y.4,887 differentially expressed genes were screened between CK-M and Mut-M with 2,406 genes up-regulated and 2,481 genes down-regulated in Mut-M.And 3,795 differentially expressed genes were screened between CK-A and Mut-A with 2,488 genes up-regulatied and 1,307 genes down-regulated in Mut-A.It was identified that totally 106 genes were up-regulated and 141 genes were down-regulated in the mutant during all three developmental stages of these two test materials.Secondly,during the development process of mutant leaves,6,894 differential genes were screened between Mut-Y and Mut-A,5,659 differential genes were screened between Mut-Y and Mut-M,and 2,447 were screened between Mut-M and Mut-A.During the three developmental stages,it was observed that 262 genes were continuously up-regulated while 178 genes were continuously down-regulated.4.According to the results of transcriptome sequencing,39 gene sequences related to the metabolism of porphyrin and chlorophyll were identified,and 14 of them were significantly differentially expressed,including AahemA-1?AahemA-2?AahemC?Aahem Y?AachlH?AaCRD1?AaPOR?AaCAO and AachlP in chlorophyll biosynthesis pathway,AaPaO and AaCLH in chlorophyll degradation pathway,AahemH in heme biosynthesis pathway and AacysG and AasirB in sirohemoglobin biosynthesis pathway.Based on the above,the differential expression characteristics of 10 genes during different developmental stages were analyzed by quantitative real-time PCR on both two materials.The results showed that the trend of expression changes was consistent with the transcriptome sequencing analysis,which verified the reliability of transcriptome sequencing data5.With comprehensive analysis,it can be concluded that the inhibition of chlorophyll biosynthesis in both young and mature leaves of the leaf color mutant occurred in the transformation process from Uroporphyrinogen ? to protoporphyrin ?,While the old leaves of the leaf color mutant occurred in the transformation process from Uroporphyrinogen ? to coproporphyrinogen ?.This suggested that the yellow leaf mutant was a total chlorophyll-deficient mutant of chlorophyll. |
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