Cotton PPR Gene Analysis And VIGS Functional Verification

Cytoplasmic male sterility(CMS) is a widespread trait in flowering plants specified by novel, often chimeric genes in the maternally inherited mitochondrial genome. The trait can be suppressed by nuclear restorer of fertility(Rf) genes. Most characterized nuclear restorer genes have been found to encode PPR proteins. The preliminary study of the experiment found that Rf1 was located on the chromosome 9 in the sequenced D5 genome of G. raimondii. This study focuses on functional analysis and VIGS validation of the PPR genes on chromosome 9 in the D5 genome. At the same time, analysis the distribution of PPR genes in the G. arboretumi genome, G. raimondii genome and G. hirsutum genome. Mega 6.0 software was used to construct the genetic evolution tree of 89 PPR genes. According to the distance of the homologous relation, these genes were divided into 9 categories, named as L1-L9. Each of the segments was inserted into the pCLCrVA vector with higher homology, and constructed as a recombinant virus vector. F1 generation plants were inoculated with Agrobacterium mediated method, and the results were as follows:1. 586 PPR genes were predicted from the G. arboretumi genome, 589 genes were predicted from the G. raimondii genome, and 1133 genes were predicted from the G. hirsutum. genome. PPR genes was present on each chromosome and present clusters. It was inferred that 42 PPR genes were lost in the process of pollen fusion and doubling in G. arboreum and G. raimondii.2. Silencing the 89 PPR genes by the VIGS technique, 66 genes of which expression was down regulated and 23 genes expression was not reduced. L1 contains 32 genes, 27 genes were down regulated. L6 contains 27 genes, only 14 genes down regulated. When the VIGS technique was used to silence many members of the gene family, gene silencing efficiency was not consistent and select the appropriate insert for silencing efficiency has an important influence.3. Fertility survey found that L1, L2 and L6 appeared single flower sterility, semi sterile pollen and without the whole plant sterile. Combining with the relative fluorescence quantitative analysis showed that in the use of VIGS silence functional genes in plants can only partial silence rather than complete silence gene function, resulting in transgenic plants showed the fertility inconsistent or unstable, also show that PPR genes may regulate cytoplasm male sterile gene expression.4. L1 positive strains appear yellow green leaf mutant, leaf margin involute or revolute, this mutation can be recovered later. The L3 and L9 positive strains appear albino color mutation, was patchy first appeared in the vicinity of the veins, finally extends to most of the blade to maintain to the late growth stage. Paraffin section analysis of the L3 and L9 plant leaves, compared with the wild type control, the spongy tissue of the leaves of L3 and L9 plants was smaller and regular, and the cell gap was smaller. VIGS technique was used to silence the single gene, and the mutation of leaf color was found in L3-1. VIGS technique was used to silence the single gene and the mutation of leaf color was found in L9-5 and L9-6. The results indicated that L3-1, L9-5 and L9-6 might be related to the mutation of leaves, and may be involved in the synthesis of chlorophyll.5. Measurement of cotton fiber length by hand pulling method. The length of cotton fiber of L1, L3 and L6 show a significant reduction than CK. The fiber length of L7 was significantly higher than CK. The result indicating that some PPR genes may relate to fiber length and elongation. Using psRNA Target site predicted the miRNA, found that 25 genes in L1 were the target of miR7505, which indicates that miR7505 may relate to fiber length.