Cloning Of ICE1-like Transcription Factors And Assessment Of The Cold Resistance In Transgenic Rice

In view of chilling damages happening frequently for rice and the important regulationof transcription factors in plant responses to low temperature, the following studies hawe beencarried out in this study:（1） Three ICE1-like genes related cold stress, members of bHLHfamily genes, were isolated from three cold-resistance plants respectively, the proteinsequences encoded by genes were analyzed, and the protein functions were predicted.（2） Themodes of tissue expression and stress expression for ICE1-like genes were analyzed,prokaryotic expression of genes was conducted, and yeast-one-hybrid system was used tovalidate transactivation function regions of ICE1-like genes.（3） Constructing plant expressionvectors and transformation into rice, the transgenic rice lines with overexpression ofICE1-like genes were used to validate the function of the genes, and the cold-resistanceeffects and regulation modes of cold responsive pathways for the genes in transgenic ricewere evaluated and analyzed respectively, expecting to provide new candidate genes forimproving rice in cold resistance and the theoretical basis for breeding rice varieties with coldresistance. The main results are as follows:（1） One ICE1-like gene, designated as LsICE1（GenBank accession No. HQ848932）, wasisolated from lettuce by single oligonucleotide nested PCR （SON-PCR） and reversetranscription-PCR （RT-PCR）. Sequence analysis showed that the LsICE1cDNA fragmentlength was1622bp containing a full coding region of1497bp encoding498amino acidresidues. The other ICE1-like gene, designated as BcICE1（GenBank accession No.HQ902162）, was isolated from Chinese cabbage by SON-PCR and RT-PCR. The full-lengthcDNA of BcICE1was1591bp with a1494bp open reading frame （ORF） encoding497aminoacid residues. Another ICE1-like gene, designated as RsICE1（GenBank accession No.HQ891287）, was isolated from radish by in silicon cloning and RT-PCR. Sequence analysisshowed that the full-length cDNA of RsICE1was1375bp and contained a complete codingregion of1266bp, which encoded421amino acid residues. Sequence multialigmentdemonstrated that the primary structures and secondary structure of the LsICE1, BcICE1,RsICE1proteins were extremly similar to that of Arabidopsis thaliana. The C-terminal of deduced LsICE1, BcICE1, RsICE1proteins were high identity with other ICE1-like proteinsfrom different species and exhibited a typical bHLH domain, SUMO binding site and nuclearlocalization signal （NLS）. The homology tree showed that LsICE1protein was at the sameevolutionary branch with ICE1-like protein of Vitis vinifera, BcICE1protein was at the sameevolutionary branch with ICE1-like proteins of Eutrema salsugineum and Thellungiellahalophila, and RsICE1protein was at the same evolutionary branch with ICE1-like proteins ofCruciferae plants, such as Eutrema salsugineum.（2） The results showed that LsICE1, BcICE1and RsICE1genes were expressedconstitutively in the examined tissues, such as the leaves, roots and stems of lettuce, Chinesecabbage and radish. LsICE1mRNA levels were dominant in leaves of lettuce seedling,BcICE1mRNA levels were dominant in stems of Chinese Cabbage seedling, while RsICE1gene mRNA levels were dominant in roots of radish seedling. Analysis of inductionexpression indicated that the mRNA levels of LsICE1, BcICE1, RsICE1genes were firstup-regulated then down-regulated in the seedlings under the low temperature stress （4°C） andhigh salt （200mM NaCl） treatments, which proved that both low-temperature and high salteffected the expression of genes. Under100μM ABA stress, the expression of LsICE1andBcICE1was up-regulated, but such stress had little effect on the expression of RsICE1gene.In addition, by time of exposure to the7%PEG6000dehydration stress, the expression ofLsICE1, BcICE1, RsICE1genes had little change observed, suggesting that the expression ofLsICE1, BcICE1, RsICE1genes was insensitive to dehydration stress.（3） The prokaryotic expression vectors for LsICE1, BcICE1, RsICE1genes respectively,pET32a-LsICE1, pET32a-BcICE1, pET32a-RsICE1, were constructed, then they weretransferred into E.coli BL21（DE3）, recombined strains were screened, and special proteinproduct with the same molecular weight as prediction was detected by SDS-PAGE afterinduced by IPTG. Transcription activation expression vectors with LsICE1, BcICE1, RsICE1gene respectively were constructed to carry out yeast-one-hybridization. The recombinantyeast strains could grow on SD/-Trp plate, and could be dyed blue by X-Gal, suggesting thatLsICE1, BcICE1, RsICE1genes could activate the expression of downstream reporter gene ofyeast, and owned the function of transcriptional activation. （4） The over-expression vectors of LsICE1, BcICE1, RsICE1genes were constructedrespectively, then LsICE1, BcICE1, RsICE1genes were transferred into rice asmonocotyledonous model plant by Agrobacterium mediated genetic transformation system,leading to over-expression of LsICE1, BcICE1, RsICE1genes in rice. A total of47transgenicrice plants with over-expression exogenous genes were obtained by the PCR, PCR-Southernand RT-PCR. detection. Among these plants, there were14transgenic rice plants with LsICE1gene,16transgenic rice plants with BcICE1gene,17transgenic rice plants with RsICE1gene,respectively. T₀transgenic rice plants grew slower, and had a delay of growth anddevelopment period than non-transgenic rice plants. The plant height, panicle length, numberof grains per panicle, seed setting rate and1000-grain weight of transgenic rice plants hadsignificant differences from those of non-transgenic plants.（5） χ²test indicated that the Hyg-resistance segregation occurred in self-pollinated seeds（T₁generation） of33T₀rice plants. Among them, the Hyg-resistance segregations of13transgenic rice lines were not in accord with3:1which showed that target genes weretransformed to rice by not a single copy and a single site, while the Hyg-resistancesegregations of20rice lines were3:1, suggesting that target genes were transformed to riceby a single copy and a single site.（6） Low temperature recovering growth method was used to test the survival rates oftransgenic rice lines under cold stress,11transgenic rice lines showed obvious cold resistancethan non-transgenic rice lines （P<0.01）. The survival rates of them were as follows:T₁L11>T₁L7>T₁L26>T₁L2>T₁L37>T₁L21>T₁L32>T₁L27>T₁L15>T₁L30>T₁L19. In addition,the transgenic rice lines with LsICE1gene had higher survival rates than the transgenic ricelines with BcICE1gene, while the transgenic rice lines with BcICE1gene had higher survivalrates than transgenic rice lines with RsICE1gene.（7） Under low temperature stress treatment, compared to control, the transgenic lines hadlower accumulation rate of relative conductivity and malondialdehyde （MDA） content. Afterlow temperature stress for72h, the final relative electric conductivity of11transgenic ricelines with cold resistance was as follows: T₁L19>T₁L27>T₁L30>T₁L32>T₁L15>T₁L2>T₁L21>T₁L26>T₁L7>T₁L11>T₁L37, which was significantly lower than that of the non-transgenic control （P<0.01）. The final relative electric conductivity increase amplitude of11transgenicrice lines with cold resistance was as follows: T₁L19>T₁L30>T₁L27>T₁L15>T₁L32>T₁L37>T₁L21>T₁L2>T₁L7>T₁L26>T₁L11, which was significantly lower than that of thenon-transgenic control. The transgenic rice lines with RsICE1gene had higher relative electricconductivity increase amplitude than the transgenic rice lines with BcICE1gene, while thetransgenic rice lines with BcICE1gene had higher relative electric conductivity increaseamplitude than transgenic rice lines with LsICE1gene.The final malonyldialdehyde （MDA）content of11transgenic plants with cold resistance was as follows: T₁L19>T₁L30>T₁L27>T₁L15>T₁L32>T₁L21>T₁L37>T₁L2>T₁L7>T₁L11>T₁L26, which was significantly lowerthan that of the non-transgenic control （P<0.01）. The final MDA increase amplitude of11transgenic rice lines with cold resistance was as follows: T₁L19>T₁L30>T₁L27>T₁L15>T₁L32>T₁L37>T₁L21>T₁L2>T₁L26>T₁L7>T₁L11, which was significantly lower than that ofthe non-transgenic control. The transgenic rice lines with RsICE1gene had higher MDAincrease amplitude than the transgenic rice lines with BcICE1gene, while the transgenic ricelines with BcICE1gene had higher MDA increase amplitude than transgenic rice lines withLsICE1gene.（8） Under low temperature stress, LsICE1, BcICE1, RsICE1gene promoted the masssynthesis of proline in transgenic plants. The final free proline content of11transgenic ricelines with cold resistance was as follows: T₁L27>T₁L21>T₁L15>T₁L7>T₁L2>T₁L11>T₁L26>T₁L32>T₁L37>T₁L19>T₁L30, which was significantly higher than that of the non-transgeniccontrol （P<0.01）. The final free proline content increase amplitude of11transgenic rice lineswith cold resistance was as follow: T₁L27>T₁L2>T₁L15>T₁L21>T₁L7>T₁L11>T₁L26>T₁L30>T₁L32>T₁L37>T₁L19>T₁L19, which was significantly higher than that of thenon-transgenic control. The transgenic rice lines with RsICE1gene had higher prolineincrease amplitude than the transgenic rice lines with LsICE1gene, while the transgenic ricelines with LsICE1gene had higher MDA increase amplitude than transgenic rice lines withBcICE1gene.（9） Southern blot analysis showed that the target genes of transgenic rice lines withLsICE1gene （T₁L2, T₁L7, T₁L11）, transgenic rice lines with BcICE1gene （T₁L15, T₁L19, T₁L21, T₁L26） and transgenic rice lines with RsICE1gene （T₁L27, T₁L30, T₁L32, T₁L37）were transformed to rice by a single copy and a single site, which was completely in accordwith the results of χ2test. Northern blot analysis further indicated that LsICE1, BcICE1,RsICE1genes were able to express normally in11T₁rice lines.（10） Real time quantitative RT-PCR was used to analyze the expression levels of coldstress-inducible transcription factors, OsDREB1F, OsDREB1B and OsDREB1A genes, intransgenic rice lines. The expression analysis of OsDREB1F, OsDREB1B and OsDREB1Agenes showed that the effects of LsICE1, BcICE1genes on the cold resistance of transgenicrice lines were dependent on CBF/DREB1cold responsive pathway, but the effect ofRsICE1genes on the cold resistance of transgenic rice lines was independent onCBF/DREB1cold responsive pathway under the cold stress. In addition, LsICE1gene wasable to upregulate significantly the transcriptional levels of OsDREB1A gene （P<0.05）, whileBcICE1gene was able to upregulate significantly the transcriptional levels of OsDREB1Fgene （P<0.05）.