Identification and expression analysis of NAC gene family in Cerasus humilis

Abstract: 【Objective】NAC (NAM, ATAF, CUC) gene, involved in a variety of metabolic activities in plants, plays an important role in stress response. With the innovation of technology, the potential functions of the NAC genes have been excavated continuously. However, there are few reports on the NAC gene in Cerasus humilis, which has strong adaptability to various stresses. Therefore, this experiment aims to identify the ChNAC gene and to clarify the structure and function of the gene by analyzing its expression pattern under different conditions.【Methods】Based on the whole genome and transcriptome data of C. humilis, combined with a variety of online websites, this study will identify and sort out the NAC gene family of C. humilis. The evolutionary relationship, conserved motif, chromosome distribution, collinearity and promoter of the C. humilis NAC family genes were also analyzed. Taking Nongda No. 4 as a material, 200 mmol·L^-1 NaCl solution and 20% PEG6000 solution were used to simulate salt and drought stress, while 4 ℃ and 40 ℃ treatments were conducted to simulate low and high temperature stress, and 25 ℃ treatment was used as control. Five biological replicates were set for each treatment. After 48 hours, 3 leaves were collected from the middle of each plant, respectively. RNA samples were sent to the company for transcriptome sequencing. Based on the transcriptome sequencing results,expression heat map of the ChNAC genes were drawn after data screening.【Results】There are 76 members in the NAC gene family of C. humilis. Most of the ChNAC genes encode basic amino acids. Most of the ChNAC proteins are hydrophilic and unstable proteins. The ChNAC proteins are mostly located in the nucleus, and the rest are located in the cytoplasm, endoplasmic reticulum, and vesicles. The proportion of Alpha helix is 10.85%-45.05%; the proportion of extended strand is 9.69%-25.27%; the proportion of β turn is 0% -9.65%; and the proportion of random coil is 38.29%-70.60%. ChNAC gene can be divided into 17 subfamilies, mainly including OSNAC7, ANAC001, ONAC009 and other subfamilies. The number of CDS fragments of the ChNAC gene ranges from 1 to 8. Most ChNAC genes are composed of motif 2, motif 8, motif 4, motif 5, motif 3, motif 6, motif 1 and motif 7. ChNAC gene mostly exists in the Chr 5 chromosome, which has 19 family members. Eight pairs of ChNAC genes are copied from large fragments of chromosome, among which three genes, including ChNAC01, ChNAC20 and ChNAC64, are collinear with each other. The results of promoter analysis showed that ChNAC gene may play roles in the growth and development, hormone regulation and stress response of C. humilis. In short, the functions of ChNAC gene are diverse and it has different biological roles. ChNAC gene also plays a great role in the growth and development of C. humilis. In order to study the expression pattern of ChNAC gene under stress environment, naturally grown Nongda No. 4 seedlings were used as the control, and the seedlings were treated with 200 mmol · L^{- 1} NaCl solution or 20% PEG6000 solution, at 4 ℃ or 40 ℃ to simulate salt stress, drought stress, low temperature stress and high temperature stress, respectively. The expression of ChNAC49 gene significantly increased under the above stresses. Under salt stress, the expression of ChNAC49 gene increased by 477 times compared with the control, while the expression of ChNAC49 gene increased by 124 times under drought stress compared with the control. Interestingly, the expression of ChNAC49 gene increased by 28 times under low temperature stress and its expression was up- regulated by 27.95 times under high temperature treatment. The expression of ChNAC64 gene decreased most with salt stress group by 14.29%, and ChNAC17 gene’s expression decreased most with drought stress group by 15.96%. The expression of ChNAC01 gene decreased most under high temperature stress, which was only 16.01% of that with control. The expression of ChNAC11 gene decreased most under high temperature stress, which was only 26.14% of that with control. These genes may play important roles in the above four stress conditions respectively, and may help C. humilis plants to maintain the normal growth and development under stress environments. In particular, the expression of ChNAC49 gene under salt, drought and low temperature stresses had the highest amplitude, and the expression of ChNAC49 gene under high temperature stress was second, only next to that of ChNAC08. It is speculated that it may also play an important role in more stresses.【Conclusion】This study identified and analyzed the physical and chemical properties and possible functions of 76 ChNAC genes, and identified the expression pattern of each ChNAC gene under different stress conditions, which lays a theoretical foundation for further study on ChNAC genes.