| Lesion mimic mutants (LMMs) are kinds of plant mutants that can spontaneously form necrotic lesion without abiotic stress or pathogen infection, resulting in increased resistance to plant defense and constitutive gene expression. LMMs are widely found in various types of plants, and are closely involved in plant programmed cell death (PCD) and plant disease resistance. These mutants are also important plant materials in study on plant stress resistance, disease resistance and genetic engineering. Study on LMMs genes is important to deeply understanding the signal network of plant PCD and the defense system of plant to different stresses. We have got two LMMs that are named as Z1828 and Z1834. Z1828 is highly resistant to rice bacterial blight, and has a broad resistance spectrum to this disease; Z1834 shows strong resistance to rice stripe disease. We have cloned and verify by transgenes the target genes of the two mutants. The main aim of this study is to screen and functionally analyze the interactive genes of the two target genes. The main results showed below:1. Screening the candidate interactive genes. The interactive genes of the two target genes of mutant Z1828 and Z1834 are screened by using yeast two-hybrid (YTH) system. As for the mutant Z1828,4 candidate interactive genes are obtained that are named as 545、546、664-1 and 664-2, respectively. The sequences of 3 candidate interactive genes (545、664-1 and 664-2) have been further cloned. The gene 545, locating on chromosome 6 and containing 8 introns, encodes a Ferredoxin-NADP reductase.664-1 and 664-2 are two different splicing types that are derived from a same gene, and there is only 12-base-pair-difference between the two splicers. The gene 664 locates on chromosome 2, contains 6 introns, and encodes a poly adenine nucleotide binding-like protein. The coding sequence (CDS) of 664-1 contains 639 base pairs, and that of 664-2 contains 651 base pairs. The gene 546 locates on chromosome 4, contains 3 introns, and encodes a cytochrome oxidase assembly protein 1. However, we have not cloned the whole sequence of this gene yet because of its unclear start codes. As for the mutant Z1834, we did not obtain the candidate interactive genes by using the whole CDS of the target gene as the bait sequence, which is possibly because that the target gene encodes a resistance protein exerting inhibitory effect on the yeast growth. So we divided the CDS into 2 different sections for screening the candidate genes. As a result, we got a only interactive gene (named as 1-5) that locates on chromosome 6and encodes a formate dehydrogenase-like protein associated with the mitochondrion precursor.2. Verifing the candidate interactive genes. We verified the 3 cloned candidate interactive genes of mutant Z1828 by using bimolecular fluorescence complementation (BiFC) and yeast two-hybrid (YTH) systems, and all results confirmeded the interaction between the candidate interactive genes and the target genes. The method of BiFC system is the construction of fusion expression vector with YFP of target gene and interacting genes, and the gene expression in tobacco cells is mediated by agrobacterium. Confocal fluorescence shows that the interaction expression of gene 545 with its target gene appears in cell periphery; and that of gene 664-1 and 664-2 in both cell periphery and nucleus. The method of YTH system is the construction of prey and bait vectors for target and interactive genes followed by coloring on SD-4. The results showed that the colony was blue and the growth condition was good, also confirming the interaction between Z1828 target gene and its candidate interactive genes. There was no results on verifing interactive gene of Z1834 target gene by using baoth BiFC and YTH system. The colony was blue on SD-4 but the growth was inhibited, which showed no interaction. This may be caused by many aspects, and further adjusted experiments are needed.3.Verifing through transgenes and T-DNA insertion mutants. The 3 candidate interactive genes was overexpressed in mutant Z1828 and the transgenic plants was obtained. Transgenic plants of gene 545 showed the delayed time of lesion-spots appearing and the significantly less spots number and density in comparision with mutant Z1828 plants. These results indicate that the interactive gene 545 can partly recovered the phenotype of mutant Z1828. Transgenic plants of gene 664-1 and 664-2 showed no significant difference from mutant Z1828 in phenotype, which may be because that the 664 gene functions in the upper stream of the target gene regulation pathway. The T-DNA insertion mutants of the candidate interactive gene 546 showed the similar phenotype of lesion mimic as the mutant Z1828, which further confirmed the interaction between gene 546 and its target gene.4. Sub-cellular location of the candidate interactive genes. Fusion expression vectors of the candidate interactive genes with YFP were constructed and transformed into both rice protoplasts and tobacco leaf cells. The sub-cellular location of gene 545 is slightly complicated with 3 kinds of situations:1. expression only in chloroplast,2. expression in cell periphery and nucleus,3. expression in all intracellular. There is slight difference of gene 664 localization between in tobacco and in protoplast cells. The gene was expressed in nucleus and cell periphery in rice protoplast, but only in nucleus in tobacco leaf cells, which may be because that the rice gene is differentially expressed in tobacco cells. Gene 1-5, the candidate interactive gene of mutant Z1834 taget gene, was expressed almost in whole cell parts except the chloroplast. |
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