发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 请教！
发信站: The unknown SPACE (Thu Oct 24 12:38:47 2002), 站内信件

【 在 wdgca (混在北京) 的大作中提到: 】
: 偶用Yeast One-Hybrid的方法分离到几个蛋白质因子的cDNA。
: 可能是植物里和抗胁迫（寒冷，干旱...)相关的转录因子，下一步想证明它在植物体内的
: 确切功能，该怎么做呢？没头绪的说，大虾救命！

analyze the homologous genes already known from AT genome to
see what people have done. get the most promising ones for
your project.
you have to verify the subcellular localization of these proteins
(in situ, western, IHC, IF, etc). establish developmental as
well as tissue-specific expression profiles for RNA/protein.
find out whether the expression or localization of these
proteins are induced, and by what kind of conditions. if so,
as you already suggested, investigate their functions under
such specific conditions.
analyze the sequence of the protein to see whether there are
potentially important sites for localization, phosphorylation,
etc, and make a serial of mutants for them. try to overexpress
these mutants in the WT background to address the phenotypes.
develop good antibody for western, IP to study the expression
and interaction with other proteins. biochemically, a lot of
experiments could be done. you can find how people usually
study an unknown transcription factor using such tools.
i do not know very much about genetics in plant, i guess
homologous recombination is still a technical problem. but as
other folks have suggested, you can try antibody interference
(you need a good antibody), RNA interference (knockdown),
or tDNA mediated knockout (homologous?), etc. and then address
the phenotypes, particularly under the stresses.
basically, you have too much to do.


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