| Biosynthesis of chlorophyll comprises a number of challenging topics in the field of plant molecular biology. People have cloned many genes involved in the course of it. AT103 is a gene cloned from leaves of Arabidopsis thaliana, which has two conserved regions, and some homologs have been investigated in photosynthetic bacterium. Those genes have been proved to be involved in the bacteriochlorophyll synthesis, but their biochemical functions of homologs in higher plant remain to be elucidated to date.In order to determine the precise function of those homologous genes in higher plants, a new gene TAT was isolated from tobacco (Nicotiana tabacum), which encodes a protein with a leucine zipper and a [(D/E)EX2H]2 motif. By searching Genbank, its homologus genes were found in purple bacteria (Rubrivivax gelatinosus), green alga (Chlamydomonas renhardtii), rice (Oryza sativa), morning glory (Pharbitis nil) et al., and a series of its homologous genes were also cloned from wheat, cucumber, clover and other plants, which proved that genes with those characteristics were ubiquitous in microorganism and higher plant. They might form a new gene family. Sequence analysis revealed that both Leucine zipper and [EXnEXRH]2 motif existed in those proteins encoded by the gene family. The former was one of classical motifs of transcriptional factors,and might help them homodimerize; the latter was the characteristic of desaturase . A cDNA fragment of about 1000bp, which shared 78.4% similarity to AT103, was amplified from tobacco leaves with degenerate oligonucleotide primers based on the conserved sequences of AT103 and PNZIP. Further, a full-length cDNA sequence was obtained by 5'amd 3' RACE PCR. The new gene, designated as TAT (Tobacco AT103-like), was 1116bp in length, with an open reading frame of 371 amino acid, and its Genbank accession number is AY221169. Northern blot analysis indicated that accumulation of mRNA was obviously different in leaves, stems, roots and petal in tobacco, and TAT gene also was induced by cold but repressed by strong lights. Simultaneously, we also cloned some homologous DNA fragments from other higher plants, suchas wheat, clover, cucumber, etc. Those genes all had high-conserved consensus sequence [EXnEXRH]2 and leucine zipper motif, which related to their biological functions and might be a good material for evolution study.In order to determine its physical and biochemical functions of the gene family in plant development, antisense AT103 gene and TAT gene expression vector under control of the CaMV35s promoter were constructed respectively, and transgenic lines were obtained in subsequent experiment. Using antisense techniques, we transferred the antisense genes into arabidopsis and tobacco. Transgenic lines of arabidopsis died after several true-leaves appeared, and the new true-leaves were more yellowish than that of non-transgenic one. Similarly, we also got the same results in tobacco leaves. After determining the content of chlorophyll in transgenic tobacco leaves, we found lower content of chlorophyll in transgenic tobacco than that of its contrast. Northern blot suggested that the expression of tobacco TAT gene decreased markedly in trnasgenic lines. By chlorophyll fluorescence and photosynthesis assay, we found that the function of chlorophyll synthesis in transgenic tobacco was repressed greatly compared with wild type, which proved that chlorophyll-deficient resulted in the fall of photosynthesis in transgenic tobacco. We may safely conclude that TAT gene and AT103 gene, its homologues in other plants as well, may play an important role in the course of chlorophyll synthesis. According to these results, we proposed that those proteins coded by the TAT gene family maybe form a new transcriptional factor family, or presumably involve in the chlorophyll synthesis as a desaturase in homodimerization.Now, the further work is undergoing in order to understand the precise function of the gene family or protein family in plant development under different physiological...
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