Improvement Of Chilling Tolerance In Tomato (Solanum Lycopersicum) Plant By Constitutive Expression Of LeHSP100/ClpB Gene

Induction of heat shock proteins is a universal response to high temperature stress among all the organisms. The HSP100 protein family is discovered among almost all kinds of plants. Similar with the HSP60,HSP70 and HSP90 protein families, the amino acid and gene sequence are highly concervative among the members of HSP100 protein family. The HSP100 protein family homologous to the bacteria Clp chaperone/protease system is involved in the protein quality control and the processes of various physiological reactions. ClpB is a member of HSP100 protein family, which broadly exists in bacteria, yeast and plant organisms, and is crucial for the basal and acquired thermotolerance. A cDNA coding for a ClpB homolog has been cloned from tomato(Solanum lycopersicum), its molecular weight is 110kD and termed as LeHSP100/ClpB. Previous studies showed that LeHSP100/ClpB was not detected under normal conditions and induced by high temperature.Using the antisense strategy, it showed that LeHSP100/ClpB was involved in thermotolerance. In order to better understand the involvement and mechanism of LeHSP100/ClpB in the anti-environmental stress process, we introduced the LeHSP100/ClpB gene into tomato plants by Agrobacterium tumefactions-mediated. With the constitutive expression of LeHSP100/ClpB, the transgenic tomato plants exhibit stronger cold tolerance than the non-transformed ones. The results were shown as follows:1 The generation of tomatoes with constitutive expressed LeHSP100/ClpB The constructed pROKII-LeHSP100/ClpB vector was introduced into Tomato by Agrobacterium tumefactions-mediated, and the constitutive expression of LeHSP100/ClpB transgenic tomato lines was generated. Northern and Western analyses confirmed the constitutive expression of the LeHSP100/ClpB transcript: the normal expression of the LeHSP100/ClpB transcript could be examined in the transgenic tomato lines at 25℃.2 Constitutive expression of LeHSP100/ClpB improved cold tolerance of tomato lines(1) Tomato plants grown under optimal growth conditions were placed into a temperature and light-controled chamber with a temperature of 4℃and a lumination of 120μmol·m-2·s-1 for 21d before recovered in normal conditions for another 7d. In the cold treatment phase, the differences in phenotypes were not observed visually between the LeHSP100/ClpB transgenic and non-transformed lines; but in the recovery phase, the non-transformed lines exhibited severe cold stress symptoms quickly with leaves pale-green, and died after 7 days. On the contrary, the transgenic lines exhibited lighter symptoms of chilling injury and kept higher values of Fv/Fm and chlorophyll content than the non-transformed lines, which meant that the constitutive expression of LeHSP100/ClpB improved the cold and low light tolerance in tomato plants.(2) Tomato plants grown under optimal growth conditions were treated with a temperature of 4℃and a lumination of 1000μmol·m-2·s-1 for 3 hours before recovered in normal conditions. The result showed that the values of Fv/Fm of transgenic tomato lines dorped more slowly and rose faster than the nontransformed lines, which indicated that the constitutive expression of LeHSP100/ClpB improved the cold and high light tolerance of tomato plants.(3) The cold and high light treatment was carried out among the LeHSP100/ClpB antisense lines and the nontransformed lines, and it turned out that the values of Fv/Fm of antisense lines dorped more quickly and rose more slowly than the nontransformed lines, which confirmed the conclusion that LeHSP100/ClpB improved the cold and high light tolerance in tomato plants.