Effects Of α-Mannosidase On Postharvest Physiological Deterioration Of Cassava Tuberous Roots

Cassava（Manihot esculenta Crantz）is a plant belonged to genus of Euphorbiaceae.Cassava was called "the king of starch" and "underground granary"because it contains a large amount of starch in tuberous roots.It play an important role in the areas of world food security,biomass energy and food processing.However,cassava tuberous root was easy to be deterioration after harvesting for 2-3 days.It appeared brown or blue-black spots around the tuberous root and then extended to the entirely tuberous root,Call it postharvest physiological decay.This phenomenon appeared in cassava tuberous root greatly affected the exploitation and utilization of cassava,and becomes a major bottleneck restricting the development of cassava industry.α-mannose glycosidase is an important protease involved in the synthesis and metabolism of glycoproteins.It plays an important role in the formation of glycoproteins.The previous studies showed that the inhibition of α-mannanosidase activity can delay fruit ripening and softening,but the effected mechanism on cassava tuberous root PPD was not clear.To further clarify the relationship between α-mannose glycosidase and PPD in cassava tuberous root,the characters of PPD in cassava tuberous root at different time points（0 d,3 d,6 d,9 d）at room temperature after harvesting were observed and recorded.The activities of α-mannanosidase were tested at different time points including 0 d,3 d,6 d and 9 d.The gene expression of α-mannanosidase at different points were determined using qRT-PCR and N-glycation level were validated using Western blot to find the key genes from α-mannanosidase to participate the PPD processing.The over expression and CRISPR/Cas9 vectors of key genes fromα-mannanosidase were constructed,and the genetic transformation of cassava TMS60444,was verified after the transgenic plants were obtained.The function of theα-mannose glycosidase gene in the PPD process after cassava harvesting was further elaborated,so as to explore the relationship between α-mannose glycosidase and PPD for cassava tuberous roots.I will provide a theoretical basis for the development of postharvest preservation technology of cassava tuberous roots.The main results of this study are as follows:1.Using 5 protein sequences encoding Ⅰ mannose glycases and 1 protein sequence encoding Ⅱ mannose glycases in Arabidopsis as references,alignment was carried out in cassava database Phytozome.Five Ⅰ mannose glycase genes（MeMNSs）and one Ⅱ mannose glycase gene（MeGMII）were obtained in the present study.2.There were no any PPD phenomena in cassava tuberous root after harvesting for 0 d.After harvest for 3 d,cassava tuberous root appeared slight PPD phenomena;the PPD area was small,mainly distributed in the peripheral part of the tuberous root.After harvest for 6 d,the root appeared more serious PPD phenomenon,and black spots appeared in the inner circumference of the tuberous root,which accounted for about one-third of the total section of the root.After harvest for 9 d,the root appeared serious PPD phenomenon,the cross section of the whole root showed black patches,and because of the loss of water,the cracks appeared in the tuberous root.3.The results determined from α-mannose glycosidase activity in cassava tuberous root at different time points showed that the mannose glycosidase activity increased with the raising of the PPD.The detection of N-glycosylation modification level during the PPD process showed that about 25 kD and 35 kD proteins were obvious modified by glycosylation,indicating that the cassava PPD process is regulated by protein glycosylation modification,which is one of the innovations in the present study.4.The analysis for the expression of 6 genes from α-mannose glycosidase in cassava tuberous root under PPD showed that MeMNS3-1 and MeGMII genes significantly increased with the PPD raising.The results inferred the genes of MeMNS3-1 and MeGMII may be the key genes involved in cassava PPD processes.5.The overexpression vector and CRISPR/Cas9 vector of MeMNS3-1 and MeGMII were constructed successfully and then transformed into cassava TMS60444（C3）to obtain cassava modified plant lines.The results showed that the target bands were detected as positive using hygromycin analysis.The further experiments will be carried on using primer amplification and sequencing techniques.