Transformation Study And Field Testing Of Candidate Genes And Promoters For Drought Resistance In Rice

Abiotic stresses such as drought, low temperature, and high salinity are major adverse environments affecting the growth and development of crops. Drought occurs frequently and becomes the bottleneck of agricultural development in many areas. Rice is one of the most important food crops for China and whole world, and rice production has consumed large amounts of water resource. So it is very significant and urgent to improve the drought resistance of crops, especially rice, and reduce the consumption of water in rice production. In this study, we have adopted a candidate gene approach to transform exogenous stress-responsive genes with known function and endogenous genes driven by constitutive and inducible promoters into rice. Transgenic rice families were tested for drought resistance under the natural field and PVC tubes conditions to identify genes and promoters that are useful for drought resistance improvement in rice. The main results are as following:1. The drought-inducible promoter OsLEAP was isolated from the upland rice IRAT109 (Oryza sativa L. ssp. japonica cv IRAT109), and the expression vector 1391Z-OsLEAP was constructed by introducing OsLEAP promoter into the vector 1391Z. GUS activity assay indicated that the activity of this promoter is strongly induced by drought, high salinity and hormone ABA, but not by cold stress.2. Three backbone vectors, 1301S (containing constitutive promoter CaMV35S), 1301A (containing constitutive promoter Actinl), and 1301H (containing drought-inducible promoter OsLEAP), were constructed on the basis of the vector pCAMBIA1301.3. Thirteen over-expression constructs for rice endogenous genes (BI119L07, BI153M13, BI133C15, BI77H21, EI77D14) were constructed on the basis of the backbone vector 1301S, 1301A, 1301H and pD1301S. Seventeen over-expression constructs for seven exogenous genes (CBF3, NCED1, SOS2, LOS5, NHX1/2, NPK1, ZAT10) were constructed on the basis of vectors pCB2005 (containing SalT promoter), pCB2006 (containing Actinl promoter), pCB2009 (containing OsHVA22 promoter). More than four thousands of transformants, about 100 transformants for each construct on average, were produced by Agrobacterium-mediated transformation using the rice cultivar Zhonghuall (Oryza sativa L. ssp. japonica) as receptor material.4. Transgenic plants were identified by PCR, Southern and Northern hybridizations. PCR analysis showed that about 94.5% of transformants were transgene-positive; Southern hybridization showed that 36.2%, 44.0% of transformants harbored single, low (2-3) copy transgene, respectively; and Northern hybridization showed that about 56.0% of transformants were overexpressing.5. Expression patterns of rice gene OsLEA3-1 (BI133C15) were investigated by Northern hybridization and the result showed that the expression of this gene was induced by drought, high salt, and hormone ABA, but not by cold stress, which is consistent with the GUS assay result of this gene promoter OsLEAP.6. Expression patterns of rice gene OsPR4-1 (EI77D14) investigated by real-time PCR in three different types of rice cultivars (Minghui 63, Zhonghua 11, and IRAT109) suggested that the expression of this gene was induced by drought, high salt, ABA and cold stresses in three cultivars. However, the induced expression level is stronger in the upland rice IRAT109 than in Minghui 63, and Zhonghua 11.7. Expression analysis of rice gene OsGO20-1 (BI119L07) indicated that the transcript of this gene was induced by drought, high salt, ABA and cold stress in three rice cultivars (Minghui 63, Zhonghua 11, and IRAT109), but not as strong as that of gene OsPR4-1. The expression of this gene was stronger in Minghui 63 and IRAT109 than in Zhonghua 11.8. Root traits (mainly maximum root length) were investigated for transgenic plants growing in PVC tubes under normal growth and drought stress conditions, but no significant difference was detected between transgenic families and wild type Zhonghua 11 (WT).9. Significant yield reduction was observed in T₁ generation of most transgenic families. Therefore, relative yield (the ratio of yield of transgenic family under drought stress to that under normal growth conditions) was used as the criterion to evaluate the drought resistance of transgenic plants in drought pre-screening under the drought shed and PVC tubes conditions. Among 30 constructs tested in the field, transgenic families of 19 constructs (CaMV35S::OsLEA3-1, OsLEAP::OsLEA3-1, D35S::OsLEA3-1, CaMV35S::OsHVA22-1, OsLEAP::OsHVA22-1, CaMV35S::OsPR4-1, OsLEAP::OsPR4-1, SalT::CBF3, Actinl::CBF3, Actinl::SOS2, OsHVA22P::SOS2, SalT-.MCED1, OsHVA22P::NCED1, Actin1:LOS5, Actinl::NHX1/2, Actin1::ZAT10, OsHVA22P::LOS5, OsHVA22P::NPK1, OsHVA22P::ZAT10) showed significant higher relative yield than WT under drought stress conditions (P＜0.05). Among 20 constructs tested in the PVC tubes, transgenic families of 15 constructs (CaMV35S::OsLEA3-1, OsLEAP::OsLEA3-1, SalT::CBF3, Actin1::CBF3, SalT::SOS2, Actinl::SOS2, OsHVA22P::SOS2, OsHVA22P::NCED1, Actin1::LOS5, Actin1::NHX1/2, Actin1::NPK1, Actin1::ZAT10, OsHVA22P::LOS5, OsHVA22P::NPK1, OsHVA22P::ZAT10) showed significant higher relative yield than WT under drought stress conditions (P＜0.05).10. According to the results of drought prescreening, some promising transgenic families (normal morphology phenotype, same yield potential as WT under normal irrigation conditions, single copy and over-expression of transgene) were selected to reproduce T₃ transgenic seeds in Hainan, drought re-testing was performed for T₃ transgenic families under drought stress conditions in Wuhan using absolute yield per plant as the criterion of drought resistance. Among the 30 tested constructs, transgenic families of 13 constructs (D35S::OsGO20-1, CaMV35S::OsLEA3-1, OsLEAP::OsLEA3-1, D35S::OsLEA3-1, CaMV35S::OsHVA22-1, Actin1::CBF3, OsHVA22P::CBF3, Actin1::LOS5, Actin1::UHX1/2, Actin1::ZAT10, OsHVA22P::LOS5, OsHVA22P::NPK1, OsHVA22P::ZAT10) showed significant higher yield per plant than WT under drought stress conditions (P＜0.05).11. Taken together, 11 constructs (CaMV35S::OsLEA3-1, OsLEAP::OsLEA3-1, D35S::OsLEA3-1,CaMV35S::OsHVA22-1,Actin1::CBF3,Actin1::OS5,Actin1::MHX1/2, Actin1::ZAT10, OsHVA22P::LOS5, OsHVA22P::NPK1, OsHVA22P::ZAT10) showed significant effect on improving drought resistance both in the prescreening (using relative yield as the criterion of drought resistance) and re-testing (using absolute yield per plant as the criterion of drought resistance) experiments; 10 constructs (OsLEAP::OsHVA22-1, CaMV35S::OsPR4-1, OsLEAP::OsPR4-1, SalT::CBF3, SalT::SOS2, Actin1::SOS2, OsHVA22P::SOS2, SalT::NCED1, OsHVA22P::NCED1,Actin1::NPK1) and 2 constructs (D35S::OsGO20-1, OsHVA22P::CBF3) showed significant effect on improving drought resistance in the prescreening, re-testing experiment, respectively; furthermore, 7 constructs (OsLEAP::OsGO20-1, D35S::OsWSI724, OsLEAP::OsWSI724, Actin1::OsLEA3-1, Actin1::OsPR4-1, Actin1::NCED1, OsHVA22P:NHX1/2) showed no effect on drought resistance improvement in either experiment.