| Ananas comosus var.bracteatus is a new type of ornamental plant whose flowers,leaves and fruits have unique ornamental value.The leaves of Ananas comosus var.bracteatus are inlaid with green and white stripes.The color is beautiful and the viewing period is long.It is an important ornamental trait of Ananas comosus var.bracteatus.In-depth research on the mechanism of albino leaves can lay a foundation for improving the stability of chimeric traits,and also help to breed new varieties with colorful leaves.At the same time,chimeras are also excellent materials for the study of plant growth.The study of the physiological and metabolic characteristics of different chimeric tissues will help to reveal the mechanism of plant growth.In this study,the Virus-induced gene silencing system was established for Ananas comosus var.bracteatus.Barley stripe mosaic virus was used as a vector.AbChlH gene is not only a key gene for chlorophyll synthesis,but also an important gene for nucleocytoplasmic signal transcription,and plays an important role in regulating the growth and development of leaf chimeric tissue.Based on the analysis of the physiological characteristics of the chimeric leaves of Ananas comosus var.bracteatus,AbChlH gene was cloned and bioinformatics was analyzed.Four AbChlH genes were silenced by VIGS,the function of this gene in chlorophyll synthesis and nucleocytoplasmic signal transcription was analyzed.The research results provide a gene expression silencing technology system for the study of the gene function of Ananas comosus var.bracteatus,and provide a theoretical basis for further revealing the formation mechanism of the golden edge chimeric trait of Ananas comosus var.bracteatus and the research on the growth characteristics of the chimera.The main results are as follows:1 VIGS system was established for Ananas comosus var.bracteatus.PDS gene was selected as indicator gene,and BSMV was used as a vector.Through the screening of bacterial concentration and infection mode,when bacterial solution OD600=0.6-0.8,resuspended solution OD600≈1.0,injection infection was adopted.Under the conditions of25℃and 90%humidity,the cells were cultivated in the dark for 48 hours,then managed normally,and the transformation effect was obvious.PCR proved that the virus successfully entered and replicated in the leaves of group（P）（infected with BSMV-γ）and group（Si）（infected with BSMV-γ-PDS）.Expression analysis of the three groups showed that Ab PDS gene expression of group（Si）was much lower than group（CK）（no treatment）and group（P）.Compared with the leaves of group（CK）,the leaves of group（P）showed a slight lightening of color around the infestation and remained green overall,while the leaves of group（Si）showed a diffuse albino trait from the infestation.The color content analysis confirmed that the chlorophyll and carotenoid contents of leaves in group（Si）were lower than those in group（CK）and group（P）.In conclusion,the VIGS system based on BSMV of Ananas comosus var.bracteatus was successfully constructed.Target gene expression is efficiently silenced and causes leaf phenotypic changes.2 A ChlH gene was identified in the genome of Ananas comosus var.bracteatus,but 4transcript sequences of AbChlH gene were cloned from both green and white tissues,named as T1、T2、T3、T4.The ORF sequences of the four genes were 4170 bp,4167bp,3324 bp,and 3321 bp,encoding 1389、1388、1107、1106 amino acids.The homology of the four amino acid sequences was 89.49%,and the homology with the magnesium chelatase of Ananas comosus、Elaeis guineensis、Phoenix dactylifera and some other plants was above 80%.It indicates that the AbChlH gene was successfully cloned.The amino acid sequences of T2 and T4 have a serine deletion at position 41 and differ from the other two sequences at five sites（113-C,R;304-D,G;894-T,A;970-M,K;1076-T,P）.In addition,T3 and T4 have intron-reserved alternative splicing,which leads to premature termination of translation of the two transcripts,and the two amino acid sequences lack 93amino acids from positions 1106 and 1105,respectively.Thus,T1 and T2 proteins have PLN03069 superfamily domains and similar protein 3D structures,may have similar functions.While the domains of T3 and T4 are incomplete,and the 3D structures of these two proteins are different to those of T1,T2 and some other species.It is speculated that T3and T4 may have lost their functions.3 The contents of chlorophyll and carotenoids in the white tissues of chimeric leaves decreased significantly,and the contents of chlorophyll anabolic intermediates Proto IX,Mg-Proto IX and Pchlide also decreased significantly.Magnesium chelatase is a key enzyme that catalyzes the formation of magnesium Mg-Proto IX from Proto IX in the plant chlorophyll synthesis pathway.It contains three catalytic subunits,namely:Chl I,Chl D,and ChlH.In the absence of catalytic substrates in the white tissue of leaves,the expression levels of the three subunits were up-regulated in white tissues,but magnesium chelatase activity was not significantly different from green tissues.4 When the AbChlH gene was silenced,the expression of AbChl I and AbChl D genes decreased,indicating that a synergistic expression relationship between AbChlH gene and AbChl I and AbChl D genes in Ananas comosus var.bracteatus.There was no significant difference in the content of Proto IX between the transgenic plants and the control plants,while the contents of chlorophyll,Mg-Proto IX and Pchlide in the leaves of the transgenic plants were significantly lower than those of the leaves of the transgenic plants.This indicated that when the AbChlH gene was silenced,the conversion of Proto IX to Mg-Proto IX was blocked,resulting in blocked chlorophyll synthesis and leaves chlorosis and whitening.5 Chlorophyll synthesis and chloroplast development were blocked in the white tissues of the chimeric leaves of Ananas comosus var.bracteatus,but the expression level of Lhcb was still similar to that in the green tissues,indicating the phenomenon of Genomes Uncoupled（GUN）.ChlH is an important gene for nucleocytoplasmic signaling.The content of Mg-Proto IX in white tissue of chimeric leaves was significantly reduced,but the expression level of AbChlH gene,magnesium chelase activity and Lhcb gene expression level were not significantly different from those in green normal tissue;When the AbChlH gene was silenced,the activity of magnesium chelatase decreased,the content of Mg-Proto IX decreased,and the expression level of Lhcb also decreased significantly,indicating that the content of Mg-Proto IX was not significantly correlated with the expression level of nuclear gene Lhcb,which may be not a signaling molecule of the nucleocytoplasmic signaling pathway.The expression level of AbChlH gene or magnesium chelatase activity may play a role in nucleocytoplasmic signaling. |
