| Cassava(Manihot esculenta Cranz)is a shrub perennial plant of Manihot belonging to Euphorbiaceous.It is an important food,feed and energy crops.Since cassava is mainly cultivated in tropical and subtropical regions between the Tropic of Tropic of Cancer Capricorn,it has adapted to warm climate and is sensitive to chilling during long-term evolution.With the increasing demand for cassava products and the rapid development of cassava industry,transplanting it to the north to expand cassava planting area is one of the most effective means to alleviate the shortage of cassava products,which puts forward requirements for the chilling tolerance of cassava varieties.Studying the molecular regulation mechanism of cassava response to chilling stress can lay a foundation for cultivating novel chilling resistant cassava varieties.In this study,MeMYBs candidate genes response to chilling stress were screened by transcriptomic data and qRT-PCR to analyze the expression patterns of MYB transcription factor family members in cassava.RNAi cassava transgenic lines of MeMYB2 were obtained by genetic transformation system.Therefore,the MeMYB2-mediated molecular mechanism of cassava in response to chilling stress was discussed based on the difference between wild-type cassava(cv.60444)(chilling-sensitive)and MeMYB2-RNAi lines.The main results were as follows:1.In this study,a total of 299 MeMYBs transcription factor family members were screened and divided into four subfamilies: R2R3-MYB,1R-MYB/MYB-related,3R-MYB and 4R-MYB.125 MeMYB genes showed different expression patterns in leaves,petioles and roots of SC124(Domestic breeding variety)and Arg7(Imported chilling resistant variety).However,the expression patterns of 142 MeMYB genes were consistent in both cassava varieties.MeMYB2 responded to chilling stress in SC124,but not in cv.60444(Transgenic Acceptors,chilling-sensitive).2.Transgenic cassava has stronger chilling tolerance.Two independent and single-copy MeMYB2 gene silencing transgenic lines 2i-17 and 2i-202 were developed by agrobacterium-mediated cassava genetic transformation system.Phenotypic analysis under chilling(4°C)stress revealed that the survival date of the transgenic lines was significantly higher than wild-type cassava.The Relative conductance(RC),contents of Malondialdehyde(MDA),H2O2 and Oxygen free radical(O2-)in RNAi transgenic cassava leaves were significantly lower than wild-type under early chilling stress.The photochemical efficiency of photosystem II(Fv/Fm),Proline content,activities of Superoxide dismutase(SOD),Catalase(CAT)and Ascorbate peroxidase(APX)in were significantly higher than in wild-type.There were significant differences in the accumulation patterns of Jasmonic Acid(JA)and Jasmonate isoleucine(JA-Ile)in leaves of RNAi transgenic cassava and wild-type,while no significant differences in Abscisic acid(ABA).3.Anthocyanin could effectively scavenge excess ROS and improve the chilling tolerance of transgenic cassava.Targeted metabolomics research on flavonoid and anthocyanin showed that the red metabolite significantly accumulated in the apical bud of RNAi transgenic cassava was cyanidin-3-O-glucoside(A class of anthocyanin substances),while it did not accumulate in the wild-type cassava.The wild-type cassava was applied with anthocyanin(grape extract),and then treated under chilling(4°C).It was found that the apical buds in the treatment group were well protected,while in the control group,they showed different degrees of wilting or death.The RC,activities of SOD,CAT and APX of treatment group was significantly lower than control group,and the Fv/Fm and Proline content of treatment group was significantly higher than control group under chilling stress.4.MeMYB2 is a negative regulator of cassava anthocyanin accumulation under chilling stress.Transcriptome KEGG enrichment analysis of DEGs between RNAi transgenic cassava and wild-type cassava showed significant differences in MAPK signaling pathway,flavonoid biosynthesis and plant-pathogen interaction pathway.Transcriptome data analysis of anthocyanin synthesis genes(Me CHS,Me CHI,Me F3 H,Me F3’H,Me F3’5’H,Me DFR,Me ANS)and Procyanidin synthesis genes(Me LAR,Me ANR)showed that all of those genes in RNAi transgenic cassava were significantly up-regulated under early chilling stress,while all of those genes in wild-type cassava did not respond to chilling.Results of qRT-PCR showed that the expression trend was consistent with the transcriptome data.5.MeTT8 is an important regulator of anthocyanin accumulation in transgenic cassava leaves under early chilling stress.Tissue specific expression analysis showed that MeTT8 was mainly expressed in leaves.Subcellular localization revealed that MeTT8 was located in the nucleus,and sequence alignment analysis revealed that MeTT8 had a typical HLH DNA-binding domain,belonging to the b HLH gene family.MeTT8 could be significantly induced by chilling in RNAi transgenic cassava compared with wild-type cassava.Yeast-one hybrid experiment,EMSA and Luciferase Complementation Assay showed that MeMYB2 could not only directly bind to the promoter of MeTT8,but also inhibit MeTT8 expression under chilling and normal temperature.MeTT8 could not only directly bind to the promoter of Me DFR,but also significantly activate Me DFR expression.6.The expression of MeTT8 in transgenic cassava was down-regulated by virus-induced gene silencing(VIGS).The anthocyanin content in MeMYB2-RNAi-MeTT8-VIGS(2iMeTT8-VIGS)lines was significantly reduced under early chilling stress,while was significantly increased in MeMYB2-RNAi-empty vector control(2i-blank).Meanwhile,the expression of Me DFR was significantly up-regulated in 2i-Blank lines,and it was inhibited in 2i-MeTT8-VIGS lines.In conclusion,after down regulating the expression of MeMYB2,chilling can induce the expression of MeTT8.MeTT8 could activate the expression of Me DFR to accumulate anthocyanins to scavenging excess ROS,so as to increase the chilling tolerance of transgenic cassava MeMYB2-RNAi.This study revealed the mechanism of chilling sensitivity of cassava to a certain extent,and created the transgenic germplasm of MeMYB2-RNAi cassava with strong chilling tolerance,which provided theoretical support for the northward transplanting of cassava. |
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