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Expression and Functional Analysis of the LeVDE in Tomato under Chilling Stress

Author: HanZuo
Tutor: MengQingWei
School: Shandong Agricultural University
Course: Botany
Keywords: Tomato High light stress Low temperature stress Violaxanthin cyclooxygenase Xanthophyll cycle Photoinhibition
CLC: S641.2
Type: PhD thesis
Year: 2009
Downloads: 291
Quote: 0
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Abstract

Light as the only energy plant photosynthesis is the basis of the plant life activities. However, when plants absorb light energy than required for electron transfer, the excess energy will cause a reduction in photosynthetic efficiency and photosynthetic function, causing photoinhibition severe photooxidative damage will occur. Plants in addition to bright light photoinhibition under low temperature and low light, high temperature, drought, salinity and other adversity due to the fixation of CO2 is limited, making the plants reduced photoinhibition of light use. Higher Plants there are a variety of defense mechanisms to avoid or reduce photoinhibition and xanthophyll cycle is considered to be an important mechanism of adversity protect the the plants photosynthetic apparatus against excess light damage. The catalyzed the xanthophyll cycle refers to the excess light energy absorbed by plants, cyclooxygenase (VDE) diepoxide violaxanthin (V) Violaxanthin anther yellow substance after intermediate single epoxy (A) is converted to the a no epoxy corn yellow mass (Z); zeaxanthin, the role of cyclooxygenase (ZE), in the dark, in the opposite direction, the Z epoxy into V, form a cycle. Non-photochemical quenching (NPQ) depends on the xanthophyll cycle to protect the plant photosynthetic apparatus from excess excitation energy damage. Excess light energy that exists Deepoxidase violaxanthin (V) and accumulated zeaxanthin (Z) is generally considered to play a crucial role in the protection of the photosynthetic apparatus. In this study, isolated from tomato leaves tomato violaxanthin off the cyclooxygenase gene and analysis of the gene expression and function. The results indicate that the expression of the gene and by light, temperature and circadian rhythm. Overexpression of this gene can reduce the tomato plants Photionhibition sensitivity, and suppression of the gene expression of tomato plants under low temperature and low light stress can aggravate photoinhibition. The main results are as follows: using homologous sequences degenerate primers by RT-PCR method from tomato leaves in the middle of the cyclooxygenase gene fragment was cloned into Violaxanthin by 5'-RACE and 3'-RACE cloned into the 5 'and 3' fragments, primers were designed to the full-length cDNA was amplified after splicing. Named LeVDE (FJ648424). The gene is 1670bp, ORF of 1437bp, encoding 478 amino acids, a molecular weight of about 53 kDa. Homologous sequences, tomato purple zeaxanthin decyclizing the the violaxanthin dismutase gene sequence of Arabidopsis, tobacco, spinach, lettuce, rice decyclizing dismutase gene sequence homology. Structural homology analysis showed LeVDE three characteristic domains, block I - terminal cysteine-rich region, the activity of VDE in the N-terminal containing 11-13 cysteine, VDE inhibitor DTT the role of region, the low concentration of DTT only partially inhibited the VDE activity, indicating that the N-terminal cysteine ??is formed more than one disulfide bond. block II lipocalin feature area, combined and transit in the region of the hydrophobic small molecule, as hydrophobic A and Z the binding region. Block III is a C-terminal glutamic acid-rich region, containing a large number of negative charges, and is the binding site of VDE and thylakoid membranes. Northern blot analysis showed that LeVDE gene was non-specific expression of high expression levels in the organization of high chlorophyll content. The same time, the gene are present in the 24-hour circadian cycle of weak light and glare treatment similar bimodal expression pattern, and in part by the inhibition of the low temperature. Southern blot analysis showed LeVDE single copy of the gene in the tomato genome. 3 would obtained LeVDE gene pBI121 vector containing the 35S promoter reorganization, the sense and antisense expression vectors were constructed, tomato leaf disk transformation using Agrobacterium-mediated method using PCR and Northern blotting with card resistant transgenic tomato plants further testing, the results proved to be successful tomato plants turn the sense and antisense gene. Compared with the wild-type plants, tomato leaves the overexpression LeVDE gene increase in the content of the A and Z, while the decrease in the content of V the off the epoxidized status than wild-type high. V amount accumulated in tomato plants LeVDE gene expression was inhibited, while the very low levels of A and Z, de-epoxidation state to maintain a lower level. Constructed prokaryotic expression vector pET-LeVDE and expressed in E. coli BL21 fusion protein will strongly induced band was cut, was dissolved in PBS to obtain antigen, immune mice antiserum titer of 1: 500. Western blot analysis showed that, in turn justice plants LeVDE gene overexpression at the protein level. In addition, chilling under low light and bright light treatment of wild-type tomato Western blot analysis. The results showed that the expression at the protein level, LeVDE always at a stable level, independent of the influence of light intensity and temperature. In the low-temperature low-light (4 ° C 100μmol m ^{-2 the s ^{-1 ) and glare (1200μmol m ^{-2 s ^{-1 ) stress conditions, wild-type and sense transgenic lines T1-7 and T1-10 strains NPQ and xanthophyll cycle de-epoxidation state (AZ) / (VAZ) have increased However, wild-type increase is even more apparent. Xanthophyll cycle-dependent NPQ to dissipate excess excitation energy under conditions of low temperature and poor light and high light stress, strengthen the transgenic lines than the wild-type dissipation capability. Fv / Fm reduce glare treatment process, the wild-type than sense transgenic strains and transgenic lines Fv / Fm recovered rapidly, the wild-type Fv / Fm slow recovery. Similar in bright light, the T1-7 and T1-10 strains Fv / Fm decreased in the low temperature stress process than the wild-type, and the slow recovery. This suggests that to reduce the PSII LeVDE overexpression of light degree of inhibition compared with transgenic tomato, the wild-type of the PSII reaction center, the more serious damage. The high light stress 12 h, the net photosynthetic rate of wild-type and transgenic tomato reduce very significantly, but the wild-type decreased significantly larger than the transgenic lines. This suggests that due to The overexpression of LeVDE enhanced chilling under low light and high light stress on the stability of the PSII reaction center, and so reduce the damage of the photosynthetic apparatus. Interim justice transgenic plants with wild-type oxidation state P700 in the low-temperature low-light process are reduced, and the wild-type decrease greater than the sense transgenic plants. After 12 h of treatment, T1-7, T1-10 and the wild-type O _{^{- | -}} content increased by 52.3%, 59.8% and 81.1%, H2O2 content increased by 40.1%, 42.3% and 61.3%, respectively. Increasing levels of wild-type in O2 and H2O2 content was significantly higher than the transgenic lines. In addition, in the low-temperature process, the to turn justice transgenic plants and the wild-type MDA content have increased, but less sense transgenic plants increased MDA content. 6 to inhibition LeVDE expression makes NPQ reduce excess light stress. Significant accumulation of antisense plants V, Z and A very low levels. Antisense plants in bright light and low-temperature low-light stress, de-epoxidation state always maintain a lower level. NPQ antisense plants in bright light and low temperature stress process in wild type and antisense plants the NPQ are rising, and 12 h after stress is lower than the wild-type. This indicates that inhibit the generation of Z so dependent on the xanthophyll cycle energy dissipation weakened. 7. Fv / Fm under high light stress of wild-type and antisense plants are reduced, but antisense reduction greater than wild-type plants. The low temperature under low light stress wild-type and antisense plants Fv / Fm decreased antisense plants decreased more significantly after cold treatment, (-) 2 (-) 9 and wild-type plants Fv / Fm decreased 20.1%, 17.9%, 12.1%. Antisense transgenic plants with wild-type oxidation state P700 reduced transit in the low-temperature low-light process, the antisense strains plant P700 decline more significant. Cold treatment (-) 2 (-) 9 and wild-type plants O ₂ ^{- | - content increased by 79.1%, 76.4% and 62.7%, respectively. After Low Light, wild-type and turn antisense gene plants MDA content, the antisense plants MDA content increased more significantly, cold stress 12 hours after the wild-type (-) and (-) 9 MDA content increased to 138.5%, 159.1% and 161.7%, respectively. In summary, the expression of LeVDE influenced by temperature, light intensity and circadian rhythms. Overexpression of the gene to reduce the sensitivity to photoinhibition of PSII and PSI. Inhibition of the expression of the gene increased photoinhibition under bright light and low-temperature low-light stress.}}}}}

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