Construction Of SSH Libraries About A Virescent Mutant From Space Mutation And Analysis Of The Differential Genes

The cotton virescent mutants exist in nature widespread. It was first found in 1925, and there are 22 mutants involved 26 virescent genes until now. The laboratory discovered a new cotton virescent mutant in the progeny of CCRI58 from space mutation, every true leaf was yellow after they emerge from the apical meristem. The main traits were as follows: the cotyledons were green, the true leaves were pale yellow when they emerged, after 3 days (the second leaf from the top) of the flattening, the etiolation phenomenon became more severely, then the leaves went to restore gradually, 10 days later (the fifth leaf from the top) the color of the mutant became normal. The previous studies generally focused on the inheritance of the virescent mutant, the molecular mechanism was still unknown. In this research, 4 SSH libraries were constructed, the forward and reverse SSH libraries of the second leaf from the top and the fifth leaf from the top of the nutant, the forward and reverse SSH libraries of the second leaf from the top of the mutant and the second leaf from the top of the wild type. The 4 SSH libraries were hoped to help us learn the cause of the the virescent phenotype. The results were as follows:1. The change of primary photochemical of different position leaves (the first leaf from the top, the second leaf from the top, the third leaf from the top, the forth leaf from the top, the fifth leaf from the top) of the wild type and the mutant were studied by chlorophyll a fluorescence transient measurement and the relevantly analytical method, JIP-test on sunny days. The value of F_v/F_m and P_IABS displayed a tendency of progressively increase in wild type, while in mutant, the second leaf from the top showed the lowest level, then increased to normal level 10 days later. The value of W_k displayed a tendency of progressively decrease in wild type, it still showed a tendency of decrease in mutant, but the young leaves (the first leaf from the top and the second leaf from the top) demonstrated extremely high. These results revealed that the the OEC of young leaves got damaged. By analyzing the parameters of M_O, S_m,φ_Eo,ψ_O in mutant, it was found that the Q_A^- accumulation was excessive in the acceptor side of PSⅡ, the electron transport chain got blocked. The change in parameters of ABS/RC, TRo/RC, ETo/RC, DIo/RC showed that the energy absorbed by reaction center was dissipated through heat and fluorescence.2. 4 SSH libraries were constructed. 4032 clones were picked and after removing the redundancy, there were 2459 Unigenes through splicing. By Blastn and Blastx, it was found that a portion of sequences involved in metabolic pathway of chlorophyll biosynthesis, the development of the chloroplast and the buliding up of photosynthetic apparatus. 14 genes were picked up to do qRT-PCR. Compared to the wild type, the relative expression of photosynthesis-related genes (PetB, PsbH, psbC, psbD and so on) transcribed by PEP was higer, and the transcribe/translation genes(rpoA and rpoB) ranscribed by NEP was lower. These results showed that the translation efficiency was low or the plastid protein was tend to degrade in the etiolation leaves of the mutant.