| Abiotic stresses such as high salinity and drought limit the normal absorption of water and nutrients from the soil by the plant,eventually leading to reduced yield,reduced quality,and even plant death.Setaria italic L.has the typical characteristics of drought resistance and barrenness resistance.Its genome(about 490 Mbp)is larger than rice(about 373 Mbp),but smaller than com(about 2300 Mbp)and wheat(about 17 Gbp),and whole genome sequencing has been completed.However,its resistance mechanism is still not clear.Therefore,overexpression of millet tolerance gene in rice to study its flinction and mechanism of action is of great significance for improving the resistance of crops.This study analyzed the characteristics and functions of the millet transcription factor genes SiMYBl,SIMYB2,and SiANTl.The main experimental contents and results are as follows:(1)The gene characteristics,promoter elements,amino acid conserved domains,and phylogenetic trees of SiMYBl,SiMYB2,and SiANTl were analyzed.The CDS of SiMYBL SIMYB2 and SiANTl genes were 915 bp,1392bp and 1863 bp,respectively.The promoters of SiMYBI and SIMYB2 genes all participated in the drought-induced MYB binding site cis-acting element MBS with the recognition sequence T(C)AACTG.All three genes have cis-acting elements ABRE that involve the abscisic acid response.SiMYBI,SiMYB2,SiANTl transcription factors have 2 SANT,1 SANT and 2AP2 amino acid conserved domains,respectively.The phylogenetic tree results show that the evolutionary relationship between millet and sorghum and com is closer.(2)The function of overexpression of SiMYBI,SiMYB2 rice was verified.The results showed that after 0.9% NaCl treatment and 10% PEG treatment,the above-ground fresh weight and dry weight of over-expressing SiMYBI rice were significantly higher than that of wild-type Kitaake except M23113-44.In fresh weight and dry weight of roots,there was no significant difference with wild type.After 10% PEG treatment,the MDA content of the three strains was significantly lower than that of the wild type.Therefore,over-expression of SiMYBl gene can improve the salt tolerance and drought tolerance of rice.Over-expression of SiMYB2 rice and wild-type rice were treated with 0.9% NaCl,and the above-ground fresh weight of SiMYB2 overexpressed rice significantly higher than that of wild-type rice.The above-ground dry weight was significantly increased except forM22943-07.After 10% PEG treatment,the MDA content of the three lines that SiMYBl overexpressed were significantly lower than that of the wild type.The fresh weight of riceaboveground and roots was significantly higher than that of the wild type except M22943-02.However,dry weights in shoots and roots were significantly higher in the three lines than inthe wild type.(3)The SiMYBl and SiMYB2 genes were cloned and the fusion expression vector wasconstructed.The subcellular localization results showed that the SiMYBl protein was in thenucleus and cytoplasm,and the SiMYB2 protein was in the nucleus.Verify that the SiMYBl transcription factor has self-activating activity.(4)The mass spectrometry analysis was performed on 0.9% NaCl stress overexpressing SiMYBl rice and wild-type Kitaake at 0,2 and 4 days,and subcellular localization of theprotein expressed by its downstream genes,construction of CRISPR vectors and overexpression vectors for downstream genes.Mass spectrometry gave a downstream proteinUCTH(ubiquitin carboxy-terminal hydrolase),UCTH protein alone in the nucleus or innucleus and cytoplasm.(5)The expression pattern of SiANTl at the seedling stage of Yugu 1 was simulated under high salinity,LN and PEG simulated drought conditions.The gene of SiANTl wasinduced by high salt stress(100 mmol-L-1 NaCl)in Yugu 1.(6)Seeds overexpressing SiANTl and wild type Kitaake were digested with 0.9% salineand tap water,respectively.The phenotypes of germinating stage and germination rate wereobserved.Rice seedlings were treated with 0.9% NaCl in Hogland nutrient solution for 10 days and then dried at 65 ° C for 48 hours,dry weights of all the rice overexpressing SiANTl and wild type(WT)plants were analyzed.At the same time,phenotype of all plantswas analyzed in the field and all plants were irrigated once with 0.9% saline before rice pregnancy.Under the similar salt treatment condition,single seed grain weigh > Plant height and tiller number per plemt of the overexpressing SiANTl rice lines and wild type were also analyzed.Under water immersion,the germination rate of SiANTl overexpression rice was similar to that of wild type.Germination rate was more than 90%.Under 0.9% saline immersion,the overexpression of SiANTl was significantly inhibited.Compared with the wild type,over-expression of SiANTl rice seeds dew white was delayed,but finally(treatmented for120h)germination rate was more than 80%.After treatment with 0.9% NaCl at seedling stagein greenhouse,the dry weight of single plant overexpressing SiANTl increased14.11%-37.42% more than that of WT,which had significant difference at the 1% level.The grain weight of transgenic rice overexpression SiANTl in field increased 56.12%-76.58%higher than that of WT,which had significant difference at the 5% level,the number of tillers per plant and plant height increased,but there was no significant difference with the wild-type.(7)It was tested whether the DNA of the SiANTl rice line was positive.Semi-quantitative RT-PCR was used to detect the expression level ?{ SiANTl over-expressed rice at RNA level.Real time-PCR method was used to analyze the relative expression of SiANTl and its possible downstream genes in transgenic rice lines.The results showed that SiANTl was expressed at the DNA and RNA level in all three overexpressing SiANTl rice lines.Real time-PCR results showed that the relative expression level of SiANTl in three rice lines overexpressing SiANTl was different but significantly increased compared with that of the recipient,and the relative expression levels of endogenous salt-tolerance-related genes OsSOSJ and OsZFP182 were 1.1-1.7 fold and1.6-2.3 fold respectively higher than that of the recipient. |
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