RESUMO
Two subtracted cDNA libraries of grapevines (Vitis vinifera. L) under short term salt stress incubation were constructed using the suppression subtractive hybridization (SSH) method combined with the differential screening approach. The mRNA isolated from leaves of the salt-tolerant grapevine cultivar Razegui grown without stress was used as a "driver," and the corresponding mRNAs isolated after a short-term treatment 6 or 24h of salt stress were used as "testers." The differentially expressed cDNA fragments were identified by differential screening of these 2 libraries. During SSH procedure, each step was operated exactly according to the manual of the kit and the results were verified correct before following step. The libraries consisted of about 7000 recombinant clones, with the average size being of 500 bp, ranging from 100 bp to 900 bp. Using a PCR-select differential screening kit, 1000 recombinant clones were randomly chosen from the subtracted cDNA libraries and hybridized with forward, reverse subtracted and unsubtracted probes for two rounds. As a result, 848 positive clones were obtained. Sequencing of randomly selected clones from the differential screening revealed that most of transcripts over-expressed by salt stress have been reported as responsive to abiotic stress response.
Assuntos
Biblioteca Gênica , RNA Mensageiro/genética , RNA de Plantas/genética , Estresse Fisiológico/genética , Vitis/genética , China , Hibridização de Ácido NucleicoRESUMO
The differential expression of PSII genes was investigated in mesophyll and bundle-sheath cells of Flaveria trinervia, a dicotyledonous C4 plant of the NADP-malic enzyme type. A comprehensive immunoblot analysis showed that three extrinsic proteins of the water-splitting complex (10, 16 and 23 kDa) are selectively depleted in mature bundle-sheath chloroplasts. In contrast, the reaction-centre core remained virtually unaffected as inferred from the abundance of the 47-kDa chlorophyll-a-binding protein, the D1 and D2 polypeptides, cytochrome b559 and the 34-kDa polypeptide. The selective depletion of the 10-, 16- and 23-kDa polypeptides in bundle-sheath chloroplasts was paralleled by a diminished PSII capacity. On the basis of oxygen evolution in the presence of the artifical electron acceptor 2,5-dimethyl-p-benzoquinone, bundle-sheath chloroplasts maintained up to 23 % of the PSII capacity shown by mesophyll chloroplasts. However, the levels of the 10-, 16- and 23-kDa proteins and, concomitantly, PSII activity varied to some degree and appeared to be correlated with environmental factors caused by seasonal changes. The selective depletion of the three members of the water-splitting complex was not reflected at the transcript level. The corresponding mRNAs were detectable in considerable amounts in bundle-sheath cells, indicating that the depletion of these proteins is regulated by post-transcriptional events. These findings reinforce the view that the peripheral proteins of the water-splitting complex are a focal point for controlling PSII activity in bundle-sheath chloroplasts of both mono- and dicotyledonous C4 plants of the NADP-malic enzyme subtype.
