Cloning, Structure Evolution And Expressing Analysis Of The Invertase Family Genes In Cassava(Manihot Esculenta Crantz)

Cassava growing in tropical and subtropical regions, and its tuber storage with rich starch, is an important food crop. Cassava is one of C3plant, but it has the highest rate of CO2fixation and sucrose synthesis. However, the accumulation of starch in roots is much lower than the theoretical yield. An increased efficiency of photo-assimilation in phloem loading, transporting, unloading and synthesizing into starch in root tuber will improve the starch yield in cassava.In higher plants, sucrose is main form of photoassimilation for transportation. In cassava phloem,80%of the saccharide is sucrose. The assimilates are photosynthesized in source organs (leaves), then are loaded-transported-unloaded to sink organs (root) through phloem, in which the sucrose metabolizing enzymes play a very important role. The invertases irreversibly decompose sucrose into glucose and fructose; meanwhile function in phloem unloading, plant growth, flowering, signal transduction, in response to biotic and abiotic stresses and so on. The invertases have several kind of isozymes, which function in photosynthesis regulation in source organs, and sink strength building in sink organs. Thus, the researches on cassava invertase gene family evolution, expression characteristics and functions can help to understand the relationship between the "source" and "sink" during the process of cassava root starch accumulation.Based on the information of cassava genome database, all cassava invertase family genes were cloned from SC8variety. The family gene evolution, gene expression characteristics, invertase activity and the content of sucrose, glucose and fructose during cassava root development were researched. The results were as follows:1、19cassava invertase genes were cloned, which are divided into three categories according to their structural characteristics:nine acid invertase genes, which including six cell wall invertases (named MeCWINV1-6, GenBank:JQ339929, JX291160, JN801147, JQ792172, JX291159, JQ339930); three vacuolar invertaes (named MeVINV1-3, GenBank: JX291158, JQ792174, JQ792173);10neutral/alkaline invertases (named MeNINV1-10, GenBank: JN616390, JQ339931, JQ339932, JQ782220, KF533729, KF533730, JN801148, JQ339933, KF533731, KF533732). The subcellular localization prediction show that MeCWINV1-6locate in cell wall; MeVINVl-3locate in vacuole; a group members of neutral/alkaline invertases locate in organelles, in which al subgroups locate in plastids and a2subgroup locate in mitochondria or plastids; β group member located in the cytoplasm.2、Based on phylogenetic analysis, subcellular localization, intron-exon and genome structure information, It is deduced that cassava acid invertase genes evolved from an original gene, which firstly evolved to form three initial genes of MeCWINV1, MeCWINV2,6and MeVINV2,2,3. MeCWINV2replicated once to form MeCWINV3, and the latter continually replicated once more to evolve MeCWINV4. The initial gene of MeCWINV2,6replicated once to form MeCWINV2and MeCWINV6. And MeCWINV2tandemly replicated to form MeCWINV5. The original gene of MeVINV1,2,3mutated to add a, vacuolar signal sequence at the N-terminal protein during evolution to form the vacuolar invertase initial gene, which replicated once to form MeVINV3and MeVINV1, and the latter replicated once more to form MeVINV2.Cassava neutral/alkaline invertase genes evolved from an original gene, which firstly evolved into a number of initial genes, al and a2subgroups evolved form respectively initial genes of MeNINV6,8and MeNINV1,7. Thee initial gene of MeNINV6,8replicated once to form MeNINV6and MeNINV8, and the latter replicated once more to form MeNINV9; the initial gene of MeNINV1,7replicated once to form MeNINV1and MeNINV7, and the latter replicated once more to form MeNINV10. The original gene evolved into β group initial genes of MeNINV4, MeNINV5and MeNINV2,3. The initial gene of MeNINV4replicated once to form nINV1, the initial gene of MeNINV2,3replicated once to MeNINV2and MeNINV3.3、The differential expression analysis of the invertase genes in tissues and organs found that:MeCWINV1mainly expresses in leaves, males and females; MeCWINV2specificly expresses in males; MeCWINV3mainly expresses in leaves and females; MeCWINV4mainly expresses in tuber root phloems and fruits; MeCWINV5mainly expresses in females and stems; MeCWINV6, MeVINV1,2mainly expresse in males, females and fruits; MeNINV1,4,7mainly expresses in stems, males and females; MeNINV6,10mainly expresses in leaves, stems, males and females; MeNINV2,3,5mainly expresses in males; MeNINV8mainly expresses in males and females; MeNINV9mainly expresses in females; nINV1mainly expresses in tuber root phloems.4、During the starch accumulation process in cassava tuber roots, MeCWINV1,3, MeVINV3, MeNINVl,6,10and nINV1were highly expressed in leaves; MeCWINV1,3, MeVINV2, MeNINVl and nINV1were highly expressed in tuber phloem; and MeCWINV1,3, MeVINV1,2, MeNINV1,6and nINV1were highly expressed in root xylem. Comprehensive analysis found that:MeCWINV1,3of cell wall invertase genes, MeCVINV2of vacuolar invertase gene and MeNINV1,6,10, nINVl of neutral/alkaline invertase genes play an important role in sucrose metabolism.5、The results from the total activity of invertase in leaves of different cassava varieties found that in leaves,91with low starch, the total activity of invertase was highest, then followed by SC124(high starch), and SC8(highest starch). It suggests that lower activity of invertase in leaves is benefit to starch accumulation in roots. The result of total activity of invertase in root phloem showed that the activity of invertase in tuber phloem of SC8and SC124was similar, which was higher than that in91. It suggests that higher activity of invertase in root phloem is benefit to starch accumulation in cassava roots. The activity of cell wall invertase was lower than that of vacuolar invertase and neutral/alkaline invertase activity in tuber, which indicats that the unloading sucrose through apoplastic pathway is lower, and it propaplly mainly transport into sink cells through plasmodesma or sucrose transporter protein.6、In initial period of tuber root formation, the sucrose content in root xylem and phloem was higher than that in leaves, it indicated that the synthesized sucrose in leaves rapidly transported to root tubers. In tuber maturity period, the sucrose content in root xylem and phloem was decreased, while it was increased in leaves, it suggests that the synthesized sucrose in leaves was inhibited to transport to root tuber as starch accumulation was slowed down in roots.In all periods of tuber root development, the content of glucose and fructose in tuber phloem and xylem was equal, which indicates that the degradation of sucrose in roots mainly through invertase. In the early period of tuber root enlargment (135days after planting), the content of glucose and fructose kept in a lower level even sucrose was in adequate level, it suggested that the rapid accumulation of starch at this period needed large amounts of glucose and fructose, which resulted in glucose and fructose were in lowest level.7、Based on the gene expression and activity of cassava invertase, subcellular localization, and sugar analysis, the probable pathway of invertase gene family involved in sucrose metabolism in cassava mesophyll cells:1, In the source organs (leaves), the triosephosphate is synthesized from the fixed CO2in calvin cycle in chloroplasts, then transported to cytoplasm through triose phosphate translocator (TPT) where sucrose is synthesized. Sucrose is transported into vacuoles, mitochondria and chloroplasts via sucrose transporter protein. Sucrose can be broken down into hexose by the neutral/alkaline invertase (nINV1, MeNINV2,3,4) and vacuolar invertase (MeVINV1,2,3) in cytoplasm or vacuole, then is phosphorylated by hexokinase to form hexose phosphate, then is re-synthesized sucrose, which is called "invalid" carbon cycle in leaves. Meanwhile, the hexose in mitochondria, sucrose is decomposed by neutral/alkaline invertase (MeNINV1,6,7,10) to produce ATP. In chloroplasts, sucrose is decomposed by neutral/alkaline invertase (MeNINV1,6,8,9), and used for starch accumulation. In cytoplasm, sucrose is decomposed UDPG and fructose by sucrose synthase. UDPG is involved in cell wall biosynthesis. Meanwhile, sucrose synthesized in leaves is transported out of the cells, then is broken down into glucose and fructose by cell wall invertase (MeCWINV1,3,4,5,6) in apoplastic space.2, in sink organs, sucrose is transported to sink cells through plasmodesmata or via sucrose transporter proteins in phloem, or is is decomposed to hexose by invertase (MeCWINV1,3,4,5,6) in cell wall apoplastic space, then transported to sink cells by hexose carriers. The sucrose in sink cells can be transported into vacuoles, mitochondria and amyloids by transporter proteins. Most of sucrose is decomposed to hexose by the neutral/alkaline invertase (nINV1, MeNINV2,3,4) in the cytoplasm and MeNINV1,6,8,9in amyloids or by the vacuolar invertase in vacuole, which is phosphorylated by hexokinase and involved in the synthesis of cassava starch. Sucrose is decomposed by neutral/alkaline invertase (MeNINV1,6,7,10) in in mitochondria to form ATP. Sucrose is decomposed to UDPG and fructose by sucrose synthase in cytoplasm, and UDPG be involved in starch and cell wall biosynthesis.