| Cotton is a major economic crop and an important raw material for the Chinese textile industry. Since grain and cotton production compete for the same land resource, there is a conflict in China with increasing population and decreasing arable land. With the wheat and cotton double cropping system, short-season cotton plays an important role in China. But the problem of earliness with decrepitude has worse effect on the yield and fiber quality of the short-season cotton. To cultivate early maturing without decrepitude cotton cultivars become a hot spot in the cotton breeding. Therefore, it is very important to clone genes about decrepitude so as to breed the improved varieties by genetically engineered methods. The main results are as follows:1. Totally 3943 clones from the normalized cDNA library were randomly selected, and 3872 readable sequences that are more than 100bp were produced with the analysis of Phrap software. The average length of the 3872 ESTs was 493bp.With the Phrap software, the 3872 effective ESTs have been analyzed by cluster and assembled into 3734 UniGene including 125 contigs and 3609 Singlets explained with composing of only one EST. Among these 3872 sequences,96.65% appear only once, 3.35% appear 2-5 times, and only one sequence appears five times. The 3872 ESTs were blasted against the non-redundant nucleotide and protein database of NCBI, and I found that an EST showed 91% homelogy with the defender against apoptotic cell death 1 (CitDAD1) in the Citrus unshiu.2. Total RNA and DNA were isolated from the leaves of CCRI 36.With RT-PCR and Chromosome Walking, GhDAD1 was cloned. The length of the gene sequence is 866bp, including a 354bp ORF,232bp 5'-UTR,280bp 3'-UTR,five extrons and 4 introns. It was in conformity with the principle of Kozak and GT-AG. Homology analysis showed that the GhDADl was highly homologous to other DAD1 from different species, especially from Populus davidiana, Citrus unshiu and Arabidopsis thaliana other than barley and rice. GhDADl protein has 112 amino acids, and its PI is 8.32. As a alkaline protein, GhDAD1 protein locates in ER.3. The RT-PCR results showed that GhDADl expressed in every tissue. The expression was higher in the young organs such as flower and embryo but lower in the old organs such as root and fibers of 20 days. QRT-PCR showed that when the cotyledon became older, with the decreasing chlorophyll, the expression levels of GhDAD1were lower.4. The CCRI10 were cultured by addition of 6-BA,ethylene,SA,NO,and H2O2.At the same time, chlorophyll was measured. The result of QRT-PCR showed that 6-BA and SA could retard the senescence, and the transcript of GhDAD1 was higher. The ethylene could accelerate the senescence, so the transcript of GhDAD1 was lower. The influence of H2O2 was not apparent. With the increasing of NO concentration, the expression level of GhDAD1increased to a peak then decreased.5. The result of fluorescence in situ hybridization showed that the signals were detected from 8 chromosomes. By RT-PCR, I found that Gossypium arboretumLand Gossypium raimondiiL contain GhDAD1 gene. Therefore both A genome and D genome have this GhDAD1 gene. But in Citrus unshiu and Arabidopsis thaliana, there are DAD families:DAD1 and DAD2.8 signals being detected showed that there might be another DAD gene in cotton. From the location of the signal, the DAD families might be on the long arm next to centromere.6. The over-expression and anti-sense RNA expressing plasmid vectors with CaMV35S PBI121-GhDADls and PBI121-GhDAD1antis were constructed using fragments of GhDAD1C.Molecular identification shows that both of the vectors along with the PBI35S(negative control)were introduced into Agobacterium tumefaciens strain LBA4404. Besides, prokaryotic expression vector constructed successfully were transformed into BL21 (DE3) strain, and induced with IPTG. The analysis of SDS-PAGE showed that there was a specific protein expression at 18.8KD molecular marker with the precipitate.It is the compound of GhDAD1 protein and His tagger.
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