Functional Research Of A20/AN1 Gene MeTIPs(2,3,5,7) In Cassava

Cassava(Manihot esculenta Crantz),also known as South American sweet potato and Manihot utilissima pohl,belongs to dicotyledonous plants.The A20/AN1-type zinc finger protein family,also known as stress associated protein(SAP)family.So far,many studies have proved that members of the A20/AN1 gene family Play a very important role in responding to abiotic stresses(such as drought,salt,cold,high temperature,heavy metals,etc.)in many plants(such as Arabidopsis thaliana,rice,banana,tobacco,etc.).However,the function and mechanism of A20/AN1 gene in cassava have not been reported.The main research results are as follows:1.In the report of the predecessor [147],14 cassava A20/AN1 genes were identified and named as MeTIP1-14.In this study,two new A20/AN1 genes were added,named MeTIP15 and MeTIP16.Except that MeTIP13,MeTIP15,and MeTIP16 only have the AN1 zinc finger domain,all other A20/AN1 genes have both an A20 zinc finger domain and an AN1 zinc finger domain.2.In this study,in order to analyze the role of MeTIPs genes in the growth and development of cassava,q PCR analysis was performed on the roots,stems,leaves,and petioles of cassva Arg7 at the four key growth stages of(1)seedling(50±5 DAP),(2)formation of root system(110±5 DAP),(3)visible root bulking(180±5 DAP),and(4) starch maturity(240±5 DAP).MeTIPs gene family is widely expressed throughout the cassava cycle,but the expression pattern is very different.3.The cassava RNA sequence data from the SRA public database was used to explore the expression of the MeTIPs genes in drought and cold.Most MeTIPs genes were up-regulated or down-regulated after drought and cold stress treatment.12 MeTIPs genes respond to drought stress and 13 MeTIPs genes respond to cold stress,respectively.MeTIPs gene may play an important role in the process of drought stress and cold stress in cassava.4.The subcellular localization of MeTIPs proteins was analyzed by transient expression in onion,tobacco and rice.MeTIPs(MeTIP1,3,4,5,8,and 11)protein is located in the nucleus,and MeTIP2 and MeTIP7 protein are located in the nucleus and cytoplasm.5.Bioinformatics analysis of MeTIP2 revealed that it is a protein with an A20 zinc finger domain and an AN1 zinc finger domain.The zinc finger domain pattern is CX(3)CX(11)CX(2)CX(2),and the AN1 zinc finger domain pattern is CX(2)CX(10)CX(1)CX(4)CX(2)HX(5)HXCX(5).There are many cis-acting elements related to abiotic and biological stress at the 1500 bp promoter sequence 5’upstream of the start site of MeTIP2 gene coding region.Phylogenetic tree and protein sequence alignment revealed that MeTIP2 protein is homologous with At SAP5,Os SAP1,Os SAP11,and Lm SAP proteins.It is suggested that MeTIP2 gene may be involved in cassava biological stress and abiotic stress.6.Compared with wild type,after 22 days of 4 % PEG6000 simulated drought stress,the root length and fresh weight of MeTIP2 transgenic plants increased by 24.85 % and 66.76 %;when grown on MS medium containing 4 % PEG6000 for 10 days,the cotyledon greening rate of MeTIP2 transgenic plants was much higher than that of wild type,which increased by 72 %;it was also found that the transgenic plants had stronger tolerance to drought stress when the 60-day potted plants of MeTIP2 were not watered for 15 days.It showed that MeTIP2 heterologous expression can improve the tolerance of transgenic plants to drought stress.It was also found that MeTIPs(MeTIP3,5,and 7)is also involved in drought stress.7.Compared with wild type,using 250 μM Al Cl3 treatment,the root length and fresh weight of MeTIP2 transgenic plants increased by38.84 % and 52.25 % on average;using 250 μM Cd Cl2 treatment,the root length and fresh weight of MeTIP2 transgenic plants increased by34.82 % and 40.79 %,respectively;under 1.5 m M Mn Cl2 treatment,there was no significant difference in root length between MeTIP2 transgenic and wild type,but the fresh weight increased by an average of 12.66 %.This indicates that MeTIP2 may play an important role in the process of resistance to heavy metal stress.8.In addition,MeTIP2 also responds to salt stress.After treatment with50 μM Na Cl,the root length of the transgenic plants did not increase significantly compared with the wild type,but the fresh weight increased significantly,with an average increase of 40.2 %.This indicates that MeTIP2 has certain biological significance in response to salt stress.In conclusion,MeTIP2,a member of the MeTIPs gene family,was preliminarily demonstrated in this study to be heterologous expression in Arabidopsis thaliana,improving Arabidopsis thaliana resistance to drought,aluminum toxicity,heavy metal cadmium,manganese stress,and salt stress.MeTIP2 and its homologues will play a very important role in developing stress-resistant cassava varieties and other crops.